Signal transduction pathway for the substance P-induced inhibition of rat Kir3(GIRK) channel
1 Department of Pharmacology
2 Department of Anatomy and Cell Biology, University of Illinois at Chicago, College of Medicine, Chicago, IL, 60612, USA
Abstract
Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP2). SP rapidly – with a half-time of 10 s with intracellular GTPS and 14 s with intracellular GTP – inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP2 than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP2. A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP2 than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP2 theory of channel inhibition. Co-immunoprecipitation reveals that Gq binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that Gq activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Gq to the channel.
, 百拇医药
Introduction
Substance P (SP), an undecapeptide belonging to the tachykinin family (Chang & Leeman, 1970), has been shown to be an excitatory transmitter in the brain and peripheral neurones (Otsuka et al. 1972). It is one of the brain transmitters in which the mechanism of slow excitation has been well investigated (Stanfield et al. 2002). The SP receptor (NK1) is located not only in the sensory tracts but also in various regions of the brain including the hippocampus, the olfactory bulb, hypothalamic nuclei and the locus coeruleus (Dam & Quirion, 1994) as well as the cholinergic neurones of the nucleus basalis. SP has been shown to inhibit inwardly rectifying K+ channels (Stanfield et al. 1985), the M-current (Nowak & Macdonald, 1982; Adams et al. 1983), or the calcium-activated K+ current (Vanner et al. 1993), thereby leading to neuronal excitation.
, http://www.100md.com
Activity of G protein-coupled inward rectifier K+ channels (Kir3; GIRK) (Dascal et al. 1993; Kubo et al. 1993) is reciprocally regulated in locus coeruleus neurones in the brain stem (Velimirovic et al. 1995): the channel is activated by somatostatin, and the same channel is suppressed by the excitatory transmitter SP. The time course of the suppression is fairly quick (a half-time of 12 s with intracellular GTPS) (Velimirovic et al. 1995). Kir3 inhibition by neurotensin in brain dopaminergic neurones is also fairly fast (Farkas et al. 1997).
, http://www.100md.com
The mechanism of Kir3 activation through G is well clarified (Logothetis et al. 1987; Reuveny et al. 1994). In contrast, the mechanism of the transmitter-induced Kir3 inhibition is poorly understood (Stanfield et al. 2002). The receptor stimulates Gq, which activates phospholipase C (PLC). PLC hydrolyses PIP2 into diacylglycerol and inositol 1,4,5-trisphosphate, resulting in a reduction of the PIP2 level. Recently, evidence has accumulated to indicate that changes in the PIP2 level regulate the activity of various ion channels (Huang et al. 1998) such as: Kir2, Kir3 (Kobrinsky et al. 2000; Cho et al. 2001; Lei et al. 2001; Meyer et al. 2001), KATP (Hilgemann & Ball, 1996; Shyng et al. 2000), KCNQ2/3 (Suh & Hille, 2002; Zhang et al. 2003), and TRP channels (Runnels et al. 2002; Prescott & Julius, 2003; Hardie, 2003).
, 百拇医药
Here, we investigated the signal transduction mechanism of Kir3 inhibition produced by SP. The main experiments have been done using the HEK293 heterologous system. We constructed mutant Kir3 and Kir2 channels in which the strength of channel binding to PIP2 was altered. The time courses of SP-induced inhibition of these mutant channels, in comparison with the wild type channels, indicate that depletion of PIP2 does not explain the SP-induced inhibition of Kir3 channels. Co-immunoprecipitation experiments have revealed that Gq interacts with Kir3.2, but not with Kir2.2 or Kir2.1. These data suggest that Gq activation quickly inhibits Kir3 channels, possibly by direct binding of Gq to the channel. A preliminary account of this work has been published (Koike et al. 2003).
, http://www.100md.com
Methods
Primary culture of brain neurones
Neurones of the nucleus basalis were cultured from 3- to 4-day-old postnatal rats as previously described (Nakajima et al. 1985; Nakajima & Masuko, 1996). The rats were anaesthetized with ether, and after the rats became completely unconscious, the forebrain was removed, immediately followed by decapitation. This protocol was reviewed and approved by the Animal Care Committee of the University of Illinois at Chicago. The removed brain was sectioned, and the nucleus basalis was isolated from brain slices under a dissection microscope. The nuclei were dissociated with papain, plated on a glial feeder layer, and cultured at 37°C with 10% CO2. The culture medium was a modified minimum essential medium with Earle's salt (88%; Gibco BRL) containing heat-inactivated rat serum (2%, prepared in our laboratory) and 50 ng ml–1 of 2.5 S nerve growth factor. The medium had been kept overnight in glia cultures (conditioned medium) (Baughman et al. 1991).
, 百拇医药
Culture and transfection of HEK293 cells
Human embryonic kidney 293 (HEK293 or HEK293A) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U ml–1) and streptomycin (100 μg ml–1) at 37°C with 10% CO2. The cells were transfected by using Effectene Transfection Reagent (Qiagen, Valencia, CA, USA). Kir3.1 and Kir3.2 cDNAs were from the rat, and G1 and G2 cDNAs were of bovine origin. SP receptor cDNA was human (Takeda et al. 1991). Mutants were constructed by a mutagenesis kit (QuikChange; Stratagene; La Jolla, CA, USA). Each mutation was verified by the sequence facility at the University of Chicago. Unless otherwise stated, cells were transfected in 6 cm culture dishes with the following cDNAs subcloned into plasmid pCMV5 (Andersson et al. 1989). To study the SP-induced effect on Kir3 channels, 0.2 μg SP receptor, 0.15 μg each of Kir3.1 and Kir3.2, 0.3 μg each of G1, and G2, and 0.1 μg green fluorescent protein (pEGFP-N1; Clontech) cDNAs per dish were used. To investigate the SP effects on Kir2.1 channels, 0.2 μg SP receptor, 0.3 μg Kir2.1 (Wischmeyer et al. 1995) and 0.1 μg pEGFP-N1 cDNAs per dish were transfected. The total amount of cDNAs was kept at 1.6 μg by adjusting the quantity of the empty vector. The next day cultures were re-plated on 3.5 cm culture dishes, which have a centre well (1.2 cm in diameter) coated with rat-tail collagen (Roche Molecular Biochem., Indianapolis, IN, USA). Electrophysiological experiments were performed 2–3 days after the re-plating.
, 百拇医药
To investigate the effects of anti-PIP2 antibody on the channels, HEK293A cells were transfected as follows. For experiments on Kir3.1/Kir3.2 (or their mutants), the standard amounts of cDNAs, unless otherwise noted, were: 0.15 μg each of Kir3.1 and Kir3.2 (or their mutants), 0.3 μg each of G1 and G2, and 0.1 μg of pEGFP-N1 with the total amount of plasmid adjusted to 1.6 μg by empty vector (for each 6 cm culture dish). To investigate Kir2.1 (or its mutants), we used 0.3 μg of Kir2.1, and 0.1 μg of pEGFP-N1, again the total amount of plasmid adjusted to 1.6 μg.
, 百拇医药
Electrophysiology
The whole-cell version of patch clamp was used. The external solution contained (mM): 141 NaCl, 10 KCl, 2.4 CaCl2, 1.3 MgCl2, 11 D-glucose, 5 Hepes-NaOH, and 0.0005 tetrodotoxin (pH 7.4). The patch pipette solution contained (mM): 141 potassium gluconate, 10 NaCl, 5 Hepes-KOH, 0.5 EGTA-KOH, 0.1 CaCl2, 4 MgCl2, 3 Na2ATP and 0.2 GTPS (or 0.2 GTP) (pH 7.2). The external solutions were exchanged with a sewer pipe manifold, except for certain early experiments, where a pressure ejector was used. The half-time of the SP-induced inhibition was obtained in most cells by a curve fitting program (Origin programs) with the Boltzmann or exponential functions.
, http://www.100md.com
The binding strength of the channels to PIP2 was determined by measuring the time courses of inactivation upon sudden application of anti-PIP2 antibody to the inside-out patch (Huang et al. 1998; Zhang et al. 1999). The patch pipette contained (mM): 155 KCl, 2.4 CaCl2, 1.3 MgCl2 and 5 Hepes(NaOH), pH 7.4. The bath solutions (bathing the cytoplasmic side) were similar to those described in Huang et al. (1998), containing fluoride (F) and vanadate (V) to inhibit the lipid phosphatases. The FV solution contained (mM): 146 KCl, 5 EGTA(KOH), 10 Hepes(KOH), 5 NaF, and 0.1 Na3VO4, pH 7.2. The FV-ATP solution contained (mM): 136 KCl, 5 EGTA(KOH), 10 Hepes(KOH), 5 NaF, 0.1 Na3VO4, 3 ATP(KOH) and 2 MgCl2 (pH 7.2). Anti-PIP2 monoclonal antibody was obtained from Assay Designs, Inc (Ann Arbor, MI, USA). The antibody was purified, thanks to the help of Stephen C. Lam and Joseph M. Schober (University of Illinois at Chicago), by protein A–sepharose. IgG2b for the negative control was from Sigma-RBI.
, http://www.100md.com
Physiological experiments were conducted at a bath temperature of 24°C. The statistical values are expressed as mean ± S.E.M.
Immunoprecipitation
HEK293 cells were transfected with plasmid cDNAs of Gq and either Myc-Kir3.2, T7-Kir2.2 or Myc-Kir2.1 by Trans IT-LT1 (Panvera). The amount of cDNA used for transfection was adjusted by adding pCMV5 to 20 μg total per 10 cm dish of the cells. Transfected cells from the 10 cm dish were harvested and solubilized in 550 μl of lysis buffer on ice for 20 min. The lysis buffer consisted of: 20 mM Hepes-NaOH at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 25 mM glycerophosphate, 1 mM Na3VO4, 2 μg ml–1 aprotinin, 10 μg ml–1 leupeptin, 10 μg ml–1 pepstatin A, 0.5% Triton-X 100, 10% glycerol and 10 μM GDP. In some experiments, 30 μM AlCl3 and 5 mM NaF were added to the lysis buffer to activate Gq. The samples were centrifuged at 15 000 g for 20 min at 4°C. The supernatants were subjected to immunoprecipitation. The cell lysates (150 μl) were incubated at 4°C for 1 h with antibody (0.3–0.5 μg) and protein A–Sepharose. Immune complexes with protein A–Sepharose were washed three times with a wash buffer (20 mM Hepes-NaOH at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5% Triton-X 100 and 10 μM GDP) either with or without 30 μM AlCl3 and 5 mM NaF. Immunoprecipitated proteins were boiled in a sample buffer (50 mM Tris-HCl at pH 6.8, 1% SDS, 2% 2-mercaptoethanol, 0.008% bromophenol blue, and 8% glycerol) and resolved by SDS-PAGE. After SDS-PAGE, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with blocking buffer (50 mM Tris-HCl at pH 8.0, 2 mM CaCl2, 80 mM NaCl, 5% skim milk, 0.2% NP-40 and 0.02% NaN3), and proteins were immunoblotted with antibodies described below. The bound antibodies were visualized by the enhanced chemiluminescence detection system (Pierce), using antimouse or antirabbit Ig antibodies conjugated with horseradish peroxidase as secondary antibodies (Amersham). Rabbit polyclonal antibody which recognizes Gq/11 was purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody 9E10 against c-Myc epitope was from Covance. Mouse monoclonal antibody against T7 epitope was from Novagen.
, 百拇医药
Results
Primary cultured brain neurones
It has been shown that sympathetic neurones can be transfected by introducing cDNAs into cell nuclei by a microinjector (Ikeda, 1997). Figure 1A–C shows that our primary cultured brain neurones from the nucleus basalis can be transfected using the same microinjection method.
A–C, primary cultured neurones from the nucleus basalis transfected by intranuclear injection of cDNA for GFP, G1 and G2. The injection pressure was 30 hPa for 0.1 s. The micrographs were taken without fixation. A, fluorescence micrograph. B and C, microscopic view of a neurone with phase-contrast in B, and fluorescence optics in C. Calibration bars: 100 μm (in A) and 50 μm (in B and C). D and E, SP-induced Kir current inhibition in nucleus basalis neurones. Injection of cDNA for RGS4 reduced the rate of SP-induced Kir inhibition. Each neurone received an intranuclear injection of cDNAs for M2-muscarinic receptors (0.3 μg μl–1), RGS4 (or empty vector) (0.5 μg μl–1), and GFP (0.1 μg μl–1). Whole-cell recordings were done 30–50 h after the injection. Holding potential, –79 mV. Command voltages of 20 mV depolarization and 50 mV hyperpolarization (each 100 ms duration) were imposed once every 4 s. The current change upon the 50 mV hyperpolarization minus the Ba2+ (100 μM)-resistant current was taken as amplitude of the Kir current. First, muscarine (20 μM) was applied to activate Kir3 channel (not shown in the figures), followed by SP (0.3 μM) application. Control: SP application caused a fast inhibition of the Kir channels (in D). A neurone injected with RGS4 cDNA showed a slow inhibition by SP (in E). F, speed of the SP-induced inhibition of the Kir channel. *P < 0.05. The diameter of the neurones was: control, 29.3 ± 1.3 μm (n = 8); RGS4, 29.8 ± 1.6 μm (n = 6). These large neurones are most probably cholinergic (Nakajima et al. 1985).
, 百拇医药
In Fig. 1D–F, cultured nucleus basalis neurones were transfected, using the microinjection method, with cDNAs for M2-muscarinic receptor, RGS4 (or empty vector for control), and GFP. We then performed whole-cell recordings using pipettes containing GTPS (200 μM). About 30 s after the giga-seal was broken, we started recording the conductance. The inward rectifier conductance (measured with 50 mV hyperpolarization) began to increase, reaching a plateau in about 5 min. This increase results from activation of endogenous Gi protein caused by spontaneous exchange of GDP (bound to Gi) with GTPS; this in turn would result in the liberation of G, which opens the Kir3 channels. In the present experiments, while the conductance was increasing, we activated the M2-muscarinic receptors by muscarine (20 μM), which further accelerated the conductance increase. After reaching a plateau, the conductance often started to decrease.
, 百拇医药
Application of SP (0.3 μM) to a control neurone after the conductance reached a plateau caused a rapid reduction of the conductance (Fig. 1D). SP receptor activation causes a rapid dissociation of Gq from the G subunit; Gq then, through an unknown mechanism, inhibits the Kir3 channels. Because the patch pipette contained GTPS, the SP application activated the receptor almost maximally without complications from receptor desensitization. The GTPS-bound G and the isolated G remain separated indefinitely.
, 百拇医药
As shown in Fig. 1E and F, the rate of the SP-induced inhibition was considerably reduced in neurones in which RGS4, an inhibitor of Gq and Gi (Hepler et al. 1997), had been additionally expressed. As there are two types of Kir channels that are affected by SP in nucleus basalis neurones (Bajic et al. 2002), the interpretation is not straightforward. Nevertheless, the present result is consistent with the notion that Gq mediates the SP-induced inhibition of native Kir3 channels in brain neurones, in agreement with conclusions from heterologous systems by Leaney et al. (2001) and Lei et al. (2001).
, http://www.100md.com
An explanation is needed on the rationale for our experimental protocol in Fig. 1D–F. The rationale relies on the difference between Gq and Gi in their activation kinetics in the presence of GTPS. Shortly after the break of the patch, the conductance begins to increase spontaneously (without receptor activation) in the presence of GTPS; this probably reflects spontaneous dissociation of Gi from G caused by an exchange of GDP with GTPS. At the peak of the conductance, activation of Gq by SP application produces a large inhibition, suggesting that the spontaneous activation of Gq by GTPS is much slower than the spontaneous activation of Gi by GTPS. Biochemical data using an in vitro preparation indicate that in order to measure the spontaneous replacement of GDP by GTPS, a 100 times higher GTPS concentration is needed for Gq than for Gi (Linder et al. 1990; Hepler et al. 1993); this means that the rate of the replacement, at the same GTPS concentration, would be much slower for Gq than for Gi.
, http://www.100md.com
RGS4 inhibits not only Gq, but also Gi. Therefore, possible inhibition of Gi by RGS4 could affect the initial spontaneous conductance increase in the presence of GTPS. Our data, however, showed that the conductance increase, which was caused by the combination of spontaneous and muscarine-induced activation, was almost the same in control and in the RGS4-transfected cells. The conductance reached a level of 203 ± 36% during the initial 254 ± 16 s in control cells (n = 8), and to a level of 220 ± 30% during the initial 304 ± 31 s in the RGS4-transfected cells (n = 8). These results indicate that the initial conductance increase was not much affected by the presence of RGS4. This is expected because RGS inhibits the G subunit only after it is split from the G, and the conductance increase of Kir3 is mediated by G not by G. By the time SP was applied, almost all the Gi had probably been fully activated in part by the spontaneous GDP–GTPS exchange, and in part by the muscarine application. Therefore, it is likely that the SP-induced conductance inhibition is caused by the Gq subunit, not by Gi, for both control and the RGS-transfected cells.
, http://www.100md.com
HEK293 cells: role of Gq
From this point on, all data were obtained from HEK293 cells. To examine the mechanism of the SP-induced Kir3 inhibition, we used HEK293 cells expressing the SP receptor, Kir3.1, Kir3.2, G1, G2 and GFP. In these cells, the Kir3.1/Kir3.2 channels were active from the beginning because of the co-transfection of G1 and G2. Application of SP to these cells produced a fairly quick inhibition (Fig. 2A). The half-time of the inhibition (T0.5) was 11 s (Fig. 2C), which is about the same as that of the SP-induced inhibition of native Kir3 channels in locus coeruleus neurones (12 s; Velimirovic et al. 1995). The N-terminus construct of G protein-coupled receptor kinase 2 (GRK2-nt) specifically interacts with the active form of Gq, but not with Gi (Carman et al. 1999). When HEK293 cells were transfected with GRK2-nt cDNA, in addition to the control set of cDNAs, the rate of the Kir3 inhibition (k0.5) became considerably slower (Fig. 2B and C), suggesting that SP inhibited the Kir3.1/Kir3.2 channels through Gq.
, 百拇医药
A, HEK293 cells transfected with SP receptor, wild type Kir3.1/Kir3.2 and G12. Application of SP (0.5 μM) inhibited the Kir3 current rapidly. Whole-cell current with the pipette solution containing GTPS (200 μM). Holding potential, –79 mV. Command voltages of 20 mV depolarization and 50 mV hyperpolarization (each 100 ms duration) were imposed once every 4 s. The amplitude of current change upon the 50 mV hyperpolarization was taken as the magnitude of the Kir current. The above protocol was used as a standard procedure in the following experiments unless otherwise stated. See Kawano et al. (1999) and Zhao et al. (2003) for the current–voltage relation recorded from HEK293 cells using a similar transfection protocol. B, rate of the inhibition (k0.5) (see text) was reduced by transfecting the cells with GRK2-nt (Carman et al. 1999), which interacts with the active form of Gq. C, comparison of the rate of SP-induced inhibition between the control (Kir3.1/Kir3.2) and the test (Kir3.1/Kir3.2 with GRK2-nt). *P = 0.0101. HEK293 cells were transfected with the standard protocol plus either 0.8 μg of empty vector (control) or GRK2-nt cDNA.
, 百拇医药
In Fig. 2, as well as in the following figures, we expressed the rate of the inhibition as the reciprocal of half-time, k0.5 = (T0.5)–1. In the present experiments, k0.5 was a better behaved variable than half-time. If half-time was used, occasionally one cell with an unusually large half-time resulted in a large arithmetic mean, overshadowing the behaviour of other cells.
Kir3 mutants
The binding strength of a channel to PIP2 is an important determinant of the channel activity (Hilgemann & Ball, 1996; Huang et al. 1998; Zhang et al. 1999). A convenient way to assess the strength of interaction is to apply anti-PIP2 antibody suddenly to the inside of the membrane and measure the rate of the conductance decrease (Huang et al. 1998). The rate is related to the ‘binding strength of the channel to PIP2’: the stronger the binding, the slower the speed of channel inactivation by the antibody (i.e. smaller value of k0.5). Affinity of the channel (reciprocal of the dissociation constant) to PIP2 can be measured by a dose–response relationship between water-soluble PIP2 and channel activity (Rohacs et al. 2002; Lopes et al. 2002; Zhang et al. 2003). Under most conditions, the two variables (the binding strength and the affinity to PIP2) would parallel each other. In fact, Logothetis' group showed that the binding strength of the channel to PIP2 measured by the antibody in various Kir2.1 mutants parallels the channel activity (Lopes et al. 2002), which in turn would represent the ‘affinity’ of the channel.
, 百拇医药
According to Zhang et al. (1999), the binding strength to PIP2 (as measured by anti-PIP2 antibody) is higher in mutant Kir3.4(I229L) than in the wild type Kir3.4. We therefore constructed Kir3.1 and Kir3.2 mutants at the residues corresponding to Kir3.4(I229L) (namely, Kir3.1(M223L) and Kir3.2(I234L)). HEK293 cells were then transfected with these mutant Kir3s. The strength of the binding of the mutant Kir3 channel to PIP2 was then examined by applying a monoclonal antibody against PIP2 to inside-out patches. As shown in Fig. 3A–C, the purified monoclonal antibody against PIP2 (50 nM) eliminated the activity of the wild type Kir3.1/Kir3.2 with a half-time of 29 s, while the same antibody treatment hardly affected the activity of Kir3.1(M223L)/Kir3.2(I234L) over several minutes (see also Fig. 3D). As a negative control, we used purified IgG2b (120 nM), which did not change the Kir3 activity (4 cells). These data, in agreement with Zhang et al. (1999), indicate that Kir3.1(M223L)/Kir3.2(I234L) has a much stronger binding to PIP2 than wild type Kir3.1/Kir3.2.
, http://www.100md.com
A and B, an inside-out patch was made from a HEK293A cell transfected with wild type Kir3.1/Kir3.2 plus G12 or Kir3.1(M223L)/Kir3.2(I234L) plus G12. Holding potential, –90 mV. The patch was first perfused with FV solution (Methods), followed by the Mg2+-ATP-containing FV solution (ATP), and finally by anti-PIP2 antibody-containing FV solution (Ab). C and D, the antibody produced a very slow inhibition in mutant Kir3.1(M223L)/Kir3.2(I234L). In C, purified monoclonal antibody was used at 50 nM. **P < 0.01. In D, a non-purified monoclonal antibody was used at 1 : 50 dilution. P = 0.06. E–G, SP-induced inhibition in whole-cell recording with pipettes containing GTPS. H–J, SP-induced inhibition with patch-pipettes containing 0.2 mM GTP. The recovery from the SP effect was measured 180 s after the peak of the SP effect: 15.5 ± 3.3% (n = 6) for the wild type and 17.8 ± 3.3% (n = 6) for the mutant. The difference is not significant.
, 百拇医药
Interestingly, the rate of SP-induced inhibition of Kir3.1(M223L)/Kir3.2(I234L) channels was essentially the same as that of the wild type Kir3.1/Kir3.2 (Fig. 3E–G). If anything, the rate of SP-induced inhibition for the mutant channels was slightly higher, in contrast to what the antibody data would suggest (Fig. 3G). This result does not agree with the idea that SP-induced Kir3 inhibition originates from depletion of PIP2 (Kobrinsky et al. 2000). If the SP-induced inhibition were caused by PIP2 depletion only, then Kir3.1(M223L)/Kir3.2(I234L), which binds strongly to PIP2, would have hardly been inhibited by the SP application.
, 百拇医药
The above experiments were all performed in the presence of internal GTPS. In contrast, the experiments in Fig. 3H–J were performed under more physiological conditions using a patch pipette solution containing GTP instead of GTPS. The result shows that the rate of SP-induced inhibition (k0.5) of the mutant channel Kir3.1(M223L)/Kir3.2(I234L) was again almost the same as that of the wild type Kir3.1/Kir3.2 (for the experiments with GTP, T0.5 was 14 s in control and 12 s in the mutant). We also measured the magnitude of conductance reduction by SP application in experiments with GTP-containing solution. The reduction was essentially the same between the wild type and the mutant; namely, for the wild type it was 51.2 ± 5.7% (n = 7) and for the mutant 49.2 ± 6.1% (n = 6). In conclusion, the results of these experiments show that the SP-induced Kir3 inhibition cannot be attributed to depletion of PIP2.
, 百拇医药
Kir2.1 (R218Q)
Kir2.1(R218Q) is a mutant with weak binding to PIP2 (Zhang et al. 1999). The channel activity of this mutant Kir2.1 therefore is readily inhibited by a small decline of the PIP2 level (Kobrinsky et al. 2000). We constructed Kir2.1(R218Q) and examined its responses to anti-PIP2 antibody and to SP application. In agreement with the result by Zhang et al. (1999), the inactivation rate by the antibody was faster in Kir2.1(R218Q) than that of the wild type Kir3.1/Kir3.2, indicating that the mutant Kir2.1(R218Q) binds to PIP2 less strongly than does the wild type Kir3.1/Kir3.2 (Fig. 4A and B).
, 百拇医药
A and B, anti-PIP2 antibody-induced inactivation of the currents recorded from inside-out patches. Holding potential, –90 mV. After activating the channel with Mg2+-ATP in FV solution, anti-PIP2 antibody (Ab) in FV solution was applied (time zero). Each symbol on the curves represents normalized mean current amplitude (n = 6). A non-purified monoclonal antibody at 1 : 200 dilution was used. *P < 0.05. C–E, SP-induced inhibition of the whole-cell current, with the pipette containing 0.2 mM GTPS. Protocol of DNA transfection was: 0.5 μg SP receptor, 0.2 μg each of Kir3.1, Kir3.2, G1 and G2 (or 1.0 μg of Kir2.1(R218Q)), and 0.1 μg of GFP (with total adjusted to 1.6 μg by pCMV5). We have sometimes noticed that SP application produced a small inward current, followed by the conductance decrease (inhibition of Kir channels) in HEK293 cells. Analysis of these transient inward currents in primary cultured neurones indicated that the transient inward current was caused by activation of non-selective cation channels (Farkas et al. 1996). We further noticed that an increase in the external divalent cation concentration tended to inhibit the non-selective cation channels. We therefore used a high Ca2+ concentration (20 mM) external solution in the hope of diminishing possible activation of non-selective cation channels (both for Kir3.1/Kir3.2 and for Kir2.1(R218Q)) (C–E), but the transient inward current still occurred. E, summary; **P < 0.01. We repeated the same experiment either with the standard conditions (Methods) or with a slight modification (internal Mg2+-ATP was 2 mM, instead of 3 mM). The overall results were: mean of k0.5 for wild type Kir3.1/Kir3.2 was 0.103 s–1 (n = 18) and that for Kir2.1(R218Q) was 0.0228 s–1 (n = 10).
, http://www.100md.com
The rates of SP-induced inhibition of these channels, however, contradicted those from PIP2 reduction. If the SP-induced inhibition was caused by PIP2 depletion, the inhibition of Kir2.1(R218Q) should occur faster than that of the wild type Kir3.1/Kir3.2. On the contrary, the time course of SP-induced inhibition of Kir2.1(R218Q) was considerably slower than that of the wild type Kir3.1/Kir3.2 (Fig. 4C–E) (P < 0.001). The result indicates that SP-induced Kir3 inhibition arises from a mechanism that is independent of PIP2 depletion.
, 百拇医药
Kir2.1
According to Zhang et al. (1999), the rank order of the binding strength of various Kir2.1 s to PIP2, as measured by the antibody, is: wild type Kir2.1 > Kir2.1(R228Q) > Kir2.1(R218Q) (Zhang et al. 1999). We examined the SP-induced inhibition of Kir2.1 channels with whole-cell recordings using patch pipettes containing GTPS. In wild type Kir2.1, we usually did not observe a SP-induced inhibition; only in 1 out of 5 cells did SP application result in a very slow inhibition. The mutant Kir2.1(R228Q) responded to SP more readily: 4 out of 7 were clearly inhibited by SP with an approximate value of k0.5 between 0.03 and 0.005 s–1. As mentioned in the previous section, the mutant Kir2.1(R218Q), which binds PIP2 weakly, showed a SP-induced inhibition with a mean k0.5 of 0.0178 s–1.
, 百拇医药
These data, although not amenable to statistical analysis, suggest that the rank order of difficulty of SP-induced channel inhibition is: Kir2.1 > Kir2.1(R228Q) > Kir2.1(R218Q). This rank order is compatible with the known order of binding strength for PIP2. Thus, as long as we consider only the responses of Kir2.1 and its mutants, the data are compatible with the idea that the SP-induced response originates from PIP2 depletion.
During this set of experiments, we also recorded the SP-induced inhibition of wild type Kir3.1/Kir3.2 (in the presence of G). The rate for Kir3.1/Kir3.2 was fast (k0.5 = 0.087 ± 0.011 s–1; n = 8) and was essentially the same as that for Kir3.1/Kir3.2 in other sets of experiments. Thus, the SP-induced inhibition rate for wild type Kir2.1 as well as that for Kir2.1(R228Q) was much slower than that of Kir3.1/Kir3.2 (in the presence of G).
, 百拇医药
The conclusion of this section is that (a) if we focus on the behaviour of Kir2.1 and its mutants, the SP-induced inhibition is compatible with the notion that inhibition originates from the depletion of PIP2, and (b) the SP-induced inhibition of Kir3.1/Kir3.2 is unique and far faster than expected from the depletion of PIP2.
Role of protein kinase C
The role of protein kinase C (PKC) in the inhibition of Kir3 channels has been a controversial subject. Using the Xenopus oocyte expression system, Sharon et al. (1997) showed that staurosporine (non-specific protein kinase inhibitor) or bisindolylmaleimide (PKC inhibitor affecting the catalytic domain) almost completely blocks the transmitter-induced inhibition of basal Kir3 current. Mao et al. (2004) showed that treatment of oocytes with calphostin C (PKC inhibitor acting on the regulatory domain) or chelerythrine (PKC inhibitor acting on the catalytic domain) completely blocks the SP-induced Kir3 inhibition (see also Hill & Peralta, 2001).
, 百拇医药
However, results obtained using mammalian host cells (HEK293) are different. Leaney et al. (2001) report that treatment with bisindolylmaleimide I or Ro-31-8220 (3 μM for at least 5 min) reduces the muscarine-induced inhibition of the basal activity of Kir3.1/Kir3.2 channels by 40–60%. Lei et al. (2001) find that application of phorbol ester produces little effect on activated (not basal) Kir3.1/Kir3.2 currents in HEK293 cells. Nor does the application of bisindolylmaleimide I (5 μM; > 30 min) have any effect on the TRH-induced inhibition of activated Kir3 channels.
, 百拇医药
We tested the effect of bisindolylmaleimide I (3 μM, for 10–30 min) in the presence of internal GTPS. As shown in Fig. 5C and D, application of the inhibitor did not change the rate (k0.5) of the SP-induced inhibition of activated Kir3.1/Kir3.2 channels (Fig. 5C). The magnitude of the SP-induced inhibition became slightly less through the application of bisindolylmaleimide I (but statistically not significant; Fig. 5D). These results agree with those of Lei et al. (2001). The most conspicuous effect produced by bisindolylmaleimide I was that the Kir3.1/Kir3.2 current itself was reduced considerably (Fig. 5E). It is interesting to note that despite the reduced conductance of Kir3.1/3.2 caused by the PKC inhibitor, the mechanism to produce the quick inhibition by SP seems mostly intact. This is in sharp contrast to the effect of the Gq inhibitor GRK-nt (Fig. 2), in which the speed of the SP-induced inhibition became slower.
, 百拇医药
HEK293 cells were transfected with cDNAs for SP receptor, Kir3.1, Kir3.2, G1 and G2 (see Methods). A, a control cell with 0.1% DMSO in the external solution. See Fig. 2 legend for the sequence of the command voltage pulses. Application of SP (1 μM) inhibited Kir3.1/Kir3.2 current. The current amplitude upon 50 mV hyperpolarization was taken as representing Kir3.1/Kir3.2 current. B, BIS-treated cells. C, the rate (k0.5) of the SP-induced inhibition was not affected by the BIS treatment (3 μM, for 11–36 min). D, the magnitude of the SP-induced Kir3.1/Kir3.2 current inhibition relative to control current. E, the BIS treatment considerably reduced the Kir3.1/Kir3.2 current (before the SP application). Ordinate: current amplitude produced by a 50 mV hyperpolarization divided by the input capacitance. *P < 0.05.
, http://www.100md.com
We can summarize the above results from different investigators as follows: when expressed in mammalian cells, the role of PKC in the receptor-mediated inhibition of strongly activated Kir3 channels seems to be small, whereas PKC does play a moderate role in inhibiting the basal activity of Kir3 channels. On the contrary, PKC does play a substantial role in the inhibition of Kir3 channels expressed in Xenopus oocytes.
Gq physically interacts with Kir3 but not with Kir2
, http://www.100md.com
The above results suggest that the SP effect on Kir3.1/Kir3.2 is faster than and of different origin from the SP effect on Kir2. There must be another quicker signalling route for Kir3.1/Kir3.2 inhibition. To elucidate the mechanism of this signalling pathway, we examined the interaction of Gq with Kir3 or with Kir2 by co-immunoprecipitation. Previously, Simen et al. (2001) reported that Gq and a type of Ca2+ channel physically interact with each other.
We found that Gq co-precipitated with Kir3.2 (Fig. 6A), whereas Gq did not co-precipitate with either Kir2.2 (Fig. 6B) or with Kir2.1 (Fig. 6C). Both the transition state form (GDP-AlF4–-bound form) and the GDP-bound form of Gq were co-precipitated with Kir3.2 (Fig. 6A), but not with Kir2.2 or Kir2.1 (Fig. 6B and C).
, http://www.100md.com
A, Gq associates with Myc-Kir3.2. (IP: Myc) Anti-Myc antibody co-precipitated Gq from cells expressing both Gq and Myc-Kir3.2 (lane 5 and lane 6). (IP: Gq) Anti-Gq/11 antibody co-precipitated Myc-Kir3.2 from cells expressing both Gq and Myc-Kir3.2. Both inactive (without AlF4–) and active (with AlF4–) forms of Gq were co-immunoprecipitated with Kir3.2. Experiments were performed using two different protocols: (i) wild type Gq with AlF4– (lane 6) or without AlF4– (lane 5), and (ii) constitutively active Gq (R183C) (without AlF4–). The results were the same for the two protocols. A representative result from 4 experiments (2 from each one of the protocols) is shown. B, Gq does not co-precipitate with Kir2.2. (IP: T7) Anti-T7 antibody did not co-precipitate Gq from cells expressing both Gq and T7-Kir2.2, while it co-precipitated T7-Kir2.2 from cells expressing T7-Kir2.2 alone (lane 4) or together with Gq (lane 5 and lane 6, top panel). (IP: Gq) Anti-Gq/11 antibody did not co-precipitate T7-Kir2.2 from cells expressing both Gq and T7-Kir2.2 (lane 5 and lane 6), whereas it co-precipitated Gq from cells expressing Gq alone (lane 2 and lane 3) and Gq with T7-Kir2.2 (lane 5 and lane 6). A representative result from 3 experiments is shown; each includes data with and without GDP-AlF4–. In addition two experimental runs were performed using Gq(R183C). These two different protocols produced the same result. C, Gq does not co-precipitate with Kir2.1. A representative result from 5 experiments is shown. * in A and C indicates IgG heavy chains of anti-Myc antibody.
, 百拇医药
Discussion
The present results show that the speed of SP-induced Kir3 inhibition was much faster than predicted from the assumption that Kir3 inhibition originates from PIP2 reduction. Although the binding strength of Kir3 to PIP2 was increased considerably (90 times) by mutation (Kir3.1(M223L)/Kir3.2(I234L)) (Fig. 3), the rate of SP-induced inhibition of the mutant channel was unchanged. As for experiments on Kir2.1(R218Q) (Fig. 4), the antibody-induced inhibition rate of Kir3.1/Kir3.2 was 2.5 times slower than that of Kir2.1(R218Q) (Fig. 4B), whereas the SP-induced inhibition of Kir3.1/Kir3.2 was 7.5 times faster than that of Kir2.1(R218Q). Hence, the SP-induced Kir3 inhibition is 19 times faster than expected from PIP2 binding characteristics. This kind of comparison, assuming a very strong affinity of the antibody to PIP2 and linear relations over large ranges, would have a considerable quantitative inaccuracy. Nonetheless, it indicates that the rapidity of the SP-induced Kir3 inhibition is by no means trivial. Thus, PIP2 reduction cannot account for the direct cause for the SP-induced inhibition of Kir3.1/Kir3.2 channels. There must be a mechanism in Kir3 channels which renders them capable of responding so rapidly to the transmitter. It is unlikely that this mechanism involves PKC. The experiments by Lei et al. (2001) and ours (Fig. 5) on the PKC inhibitor bisindolylmaleimide I indicate that, unlike the results obtained using Xenopus oocyts (Sharon et al. 1997; Hill & Peralta, 2001; Mao et al. 2004), PKC seems to play a relatively small role in the SP-induced inhibition of activated Kir3 channels expressed in mammalian host cells.
, 百拇医药
Recently, a GFP-tagged PH domain of PLC has been used as an optical probe to observe PIP2 hydrolysis (Runnels et al. 2002; Rohacs et al. 2002; Zhang et al. 2003; Suh et al. 2004). The data show that PIP2 hydrolysis by receptor activation occurs as fast as the rate of channel inhibition. These data support the notion (Suh & Hille, 2002; Zhang et al. 2003; Suh et al. 2004) that transmitter-induced inhibition of KCNQ2/3 is primarily caused by depletion of PIP2. Is our conclusion compatible with these findings A crucial factor to consider is the large variation in the PIP2 affinity among different types of channels. The PIP2 affinity of KCNQ2/3 is much less than that of Kir2.1, with an EC50 of 90 μM for KCNQ2/3, and 5 μM for Kir2.1 (EC50 is measured with a water-soluble diC8-PIP2; Rohacs et al. 2002; Zhang et al. 2003). This means that KCNQ2/3 could be inhibited by a small decline of PIP2, while Kir3 (in the presence of G) or Kir2, with its inherently high PIP2 affinity (Huang et al. 1998), may remain active even with a considerably reduced level of PIP2. Therefore, the conclusion drawn from KCNQ studies does not necessarily contradict our conclusion from Kir3 studies. Importantly, the optical experiments do not describe the relative amount of PIP2 remaining inside the membrane after transmitter application.
, 百拇医药
Previously, Huang et al. (1995) and Peleg et al. (2002) showed, using the glutathione S-transferase (GST) pull down assay, that Gi and/or G interact with the Kir3 N-terminus. Furthermore, Dascal's group (Schreibmayer et al. 1996; Peleg et al. 2002) has shown that Gi inhibits the basal Kir3 activity independent of its ability to sequester G. Thus, Gq and Gi, despite mediating opposite gross effects on Kir3 channel activity, may actually share certain signalling capabilities. The question is to explain these opposite effects on channel activity despite the fact that both Gi and Gq seem to be capable of inhibiting channel activity. Furthermore, channel-activating G is liberated regardless of the G protein system under stimulation. The functional differences may arise simply due to the quantitative strengths of the involved G protein subunits. The capability of Gq to inhibit Kir3 is strong enough to nullify the activating effect of G (Velimirovic et al. 1995), whereas channel inhibition by Gi is so weak as to be easily overcome by G.
, http://www.100md.com
We have presented two novel findings from the present research: (a) a unique mechanism must exist between Gq and Kir3.1/Kir3.2 to produce inhibition of Kir3. This mechanism does not exist between Gq and Kir2.1; (b) there is a physical interaction between Gq and Kir3.2, but not between Gq and Kir2.1 or Kir2.2. Furthermore, the literature shows that there is some similarity between Gi and Gq in their capacity to inhibit Kir3 activity (Peleg et al. 2002) and to physically interact with Kir3 (Huang et al. 1995; Peleg et al. 2002). A simple hypothetical mechanism that explains all these facts might be a signalling pathway that includes a protein–protein interaction between Gq and Kir3, resulting in closure of the channel gate.
, 百拇医药
Footnotes
M. Koike-Tani and J. M. Collins contributed equally to this work.
References
Adams PR, Brown DA & Jones SW (1983). Substance P inhibits the M-current in bullfrog sympathetic neurones. Br J Pharmacol 79, 330–333.
Andersson S, Davis DL, Dahlback H, Jornvall H & Russell DW (1989). Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem 264, 8222–8229.
, 百拇医药
Bajic D, Koike M, Albsoul-Younes AM, Nakajima S & Nakajima Y (2002). Two different inward rectifier K+ channels are effectors for transmitter-induced slow excitation in brain neurons. Proc Natl Acad Sci U S A 99, 14494–14499.
Baughman RW, Huettner JE, Jones KA & Khan AA (1991). Cell culture of neocortex and basal forebrain from postnatal rats. In Culturing Nerve Cell, ed. Baker G & Goslin K, pp. 227–249. MIT Press, Cambridge, Massachusetts.
, 百拇医药
Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedegaertner PB, Gilman AG, Benovic JL & Kozasa T (1999). Selective regulation of Galpha (q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. J Biol Chem 274, 34483–34492.
Chang MM & Leeman SE (1970). Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J Biol Chem 245, 4784–4790.
Cho H, Nam GB, Lee SH, Earm YE & Ho WK (2001). Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in alpha (1) -adrenergic pathway via the modulation of acetylcholine-activated K+ channels in mouse atrial myocytes. J Biol Chem 276, 159–164.
, http://www.100md.com
Dam T-V & Quirion R (1994). Comparative distribution of receptor types in the mammalian brain. In The Tachykinin Receptors, ed. Buck SH, pp. 101–123. Humana Press, Totowa, New Jersey.
Dascal N et al. (1993). Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc Natl Acad Sci U S A 90, 10235–10239.
Farkas RH, Chien PY, Nakajima S & Nakajima Y (1996). Properties of a slow nonselective cation conductance modulated by neurotensin and other neurotransmitters in midbrain dopaminergic neurons. J Neurophysiol 76, 1968–1981.
, http://www.100md.com
Farkas RH, Chien PY, Nakajima S & Nakajima Y (1997). Neurotensin and dopamine D2 activation oppositely regulate the same K+ conductance in rat midbrain dopaminergic neurons. Neurosci Lett 231, 21–24.
Hardie RC (2003). Regulation of TRP channels via lipid second messengers. Annu Rev Physiol 65, 735–759.
Hepler JR, Berman DM, Gilman AG & Kozasa T (1997). RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. Proc Natl Acad Sci U S A 94, 428–432.
, 百拇医药
Hepler JR, Kozasa T, Smrcka AV, Simon MI, Rhee SG, Sternweis PC & Gilman AG (1993). Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits. J Biol Chem 268, 14367–14375.
Hilgemann DW & Ball R (1996). Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2. Science 273, 956–959.
Hill JJ & Peralta EG (2001). Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor. J Biol Chem 276, 5505–5510.
, http://www.100md.com
Huang CL, Feng S & Hilgemann DW (1998). Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G beta gamma. Nature 391, 803–806.
Huang CL, Slesinger PA, Casey PJ, Jan YN & Jan LY (1995). Evidence that direct binding of G beta gamma to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation. Neuron 15, 1133–1143.
Ikeda SR (1997). Heterologous expression of receptors and signaling proteins in adult mammalian sympathetic neurons by microinjection. Meth Mol Biol 83, 191–202.
, 百拇医药
Kawano T, Chen L, Watanabe SY, Yamauchi J, Kaziro Y, Nakajima Y, Nakajima S & Itoh H (1999). Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K+ channels. FEBS Lett 463, 355–359.
Kobrinsky E, Mirshahi T, Zhang H, Jin T & Logothetis DE (2000). Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat Cell Biol 2, 507–514.
Koike M, Collins J, Kawano T, Zhao P, Zhao Q, Kozasa T, Nakajima S & Nakajima Y (2003). Signal transduction of GIRK inhibition by substance P. Biophys J 84, 226a.
, http://www.100md.com
Kubo Y, Reuveny E, Slesinger PA, Jan YN & Jan LY (1993). Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364, 802–806.
Leaney JL, Dekker LV & Tinker A (2001). Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca2+ -independent protein kinase C. J Physiol 534, 367–379.
Lei Q, Talley EM & Bayliss DA (2001). Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G (alpha) q family subunits, phospholipase C, and a readily diffusible messenger. J Biol Chem 276, 16720–16730.
, http://www.100md.com
Linder ME, Ewald DA, Miller RJ & Gilman AG (1990). Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli. J Biol Chem 265, 8243–8251.
Logothetis DE, Kurachi Y, Galper J, Neer EJ & Clapham DE (1987). The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325, 321–326.
Lopes CM, Zhang H, Rohacs T, Jin T, Yang J & Logothetis DE (2002). Alterations in conserved Kir channel–PIP2 interactions underlie channelopathies. Neuron 34, 933–944.
, http://www.100md.com
Mao J, Wang X, Chen F, Wang R, Rojas A, Shi Y, Piao H & Jiang C (2004). Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. Proc Natl Acad Sci U S A 101, 1087–1092.
Meyer T, Wellner-Kienitz MC, Biewald A, Bender K, Eickel A & Pott L (2001). Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes. J Biol Chem 276, 5650–5658.
, 百拇医药
Nakajima Y & Masuko S (1996). A technique for culturing brain nuclei from postnatal rats. Neurosci Res 26, 195–203.
Nakajima Y, Nakajima S, Obata K, Carlson CG & Yamaguchi K (1985). Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei. Proc Natl Acad Sci U S A 82, 6325–6329.
Nowak LM & Macdonald RL (1982). Substance P: ionic basis for depolarizing responses of mouse spinal cord neurons in cell culture. J Neurosci 2, 1119–1128.
, http://www.100md.com
Otsuka M, Konishi S & Takahashi T (1972). A further study of the motoneurons-depolarizing peptide extracted from dorsal roots of bovine spinal nerves. Proc Jpn Acad 48, 747–752.
Peleg S, Varon D, Ivanina T, Dessauer CW & Dascal N (2002). G(alpha)(i) controls the gating of the G protein-activated K+ channel, GIRK. Neuron 33, 87–99.
Prescott ED & Julius D (2003). A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity. Science 300, 1284–1288.
, http://www.100md.com
Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez-Lluhi JA, Lefkowitz RJ, Bourne HR, Jan YN & Jan LY (1994). Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. Nature 370, 143–146.
Rohacs T, Lopes C, Mirshahi T, Jin T, Zhang H & Logothetis DE (2002). Assaying phosphatidylinositol bisphosphate regulation of potassium channels. Meth Enzymol 345, 71–92.
Runnels LW, Yue L & Clapham DE (2002). The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat Cell Biol 4, 329–336.
, 百拇医药
Schreibmayer W, Dessauer CW, Vorobiov D, Gilman AG, Lester HA, Davidson N & Dascal N (1996). Inhibition of an inwardly rectifying K+ channel by G-protein alpha-subunits. Nature 380, 624–627.
Sharon D, Vorobiov D & Dascal N (1997). Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes. J General Physiol 109, 477–490.
Shyng SL, Cukras CA, Harwood J & Nichols CG (2000). Structural determinants of PIP (2) regulation of inward rectifier KATP channels. J General Physiol 116, 599–608.
, http://www.100md.com
Simen AA, Lee CC, Simen BB, Bindokas VP & Miller RJ (2001). The C terminus of the Ca channel alpha1B subunit mediates selective inhibition by G-protein-coupled receptors. J Neurosci 21, 7587–7597.
Stanfield PR, Nakajima S & Nakajima Y (2002). Constitutively active and G-protein coupled inward rectifier K+ channels: Kir2.0 and Kir3.0. Rev Physiol Biochem Pharmacol 145, 47–179.
Stanfield PR, Nakajima Y & Yamaguchi K (1985). Substance P raises neuronal membrane excitability by reducing inward rectification. Nature 315, 498–501.
, 百拇医药
Suh BC & Hille B (2002). Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35, 507–520.
Suh BC, Horowitz LF, Hirdes W, Mackie K & Hille B (2004). Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq. J General Physiol 123, 663–683.
Takeda Y, Chou KB, Takeda J, Sachais BS & Krause JE (1991). Molecular cloning, structural characterization and functional expression of the human substance P receptor. Biochem Biophys Res Commun 179, 1232–1240.
, http://www.100md.com
Vanner S, Evans RJ, Matsumoto SG & Surprenant A (1993). Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia. J Neurophysiol 69, 1632–1644.
Velimirovic BM, Koyano K, Nakajima S & Nakajima Y (1995). Opposing mechanisms of regulation of a G-protein-coupled inward rectifier K+ channel in rat brain neurons. Proc Natl Acad Sci U S A 92, 1590–1594.
Wischmeyer E, Lentes KU & Karschin A (1995). Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line. Pflugers Arch 429, 809–819.
, 百拇医药
Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T & Logothetis DE (2003). PIP2 activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37, 963–975.
Zhang H, He C, Yan X, Mirshahi T & Logothetis DE (1999). Activation of inwardly rectifying K+ channels by distinct PtdIns (4,5),P2 interactions. Nat Cell Biol 1, 183–188.
Zhao Q, Kawano T, Nakata H, Nakajima Y, Nakajima S & Kozasa T (2003). Interaction of G protein beta subunit with inward rectifier K+ channel Kir3. Mol Pharmacol 64, 1085–1091., 百拇医药(Maki Koike-Tani, John M C)
2 Department of Anatomy and Cell Biology, University of Illinois at Chicago, College of Medicine, Chicago, IL, 60612, USA
Abstract
Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP2). SP rapidly – with a half-time of 10 s with intracellular GTPS and 14 s with intracellular GTP – inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP2 than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP2. A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP2 than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP2 theory of channel inhibition. Co-immunoprecipitation reveals that Gq binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that Gq activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Gq to the channel.
, 百拇医药
Introduction
Substance P (SP), an undecapeptide belonging to the tachykinin family (Chang & Leeman, 1970), has been shown to be an excitatory transmitter in the brain and peripheral neurones (Otsuka et al. 1972). It is one of the brain transmitters in which the mechanism of slow excitation has been well investigated (Stanfield et al. 2002). The SP receptor (NK1) is located not only in the sensory tracts but also in various regions of the brain including the hippocampus, the olfactory bulb, hypothalamic nuclei and the locus coeruleus (Dam & Quirion, 1994) as well as the cholinergic neurones of the nucleus basalis. SP has been shown to inhibit inwardly rectifying K+ channels (Stanfield et al. 1985), the M-current (Nowak & Macdonald, 1982; Adams et al. 1983), or the calcium-activated K+ current (Vanner et al. 1993), thereby leading to neuronal excitation.
, http://www.100md.com
Activity of G protein-coupled inward rectifier K+ channels (Kir3; GIRK) (Dascal et al. 1993; Kubo et al. 1993) is reciprocally regulated in locus coeruleus neurones in the brain stem (Velimirovic et al. 1995): the channel is activated by somatostatin, and the same channel is suppressed by the excitatory transmitter SP. The time course of the suppression is fairly quick (a half-time of 12 s with intracellular GTPS) (Velimirovic et al. 1995). Kir3 inhibition by neurotensin in brain dopaminergic neurones is also fairly fast (Farkas et al. 1997).
, http://www.100md.com
The mechanism of Kir3 activation through G is well clarified (Logothetis et al. 1987; Reuveny et al. 1994). In contrast, the mechanism of the transmitter-induced Kir3 inhibition is poorly understood (Stanfield et al. 2002). The receptor stimulates Gq, which activates phospholipase C (PLC). PLC hydrolyses PIP2 into diacylglycerol and inositol 1,4,5-trisphosphate, resulting in a reduction of the PIP2 level. Recently, evidence has accumulated to indicate that changes in the PIP2 level regulate the activity of various ion channels (Huang et al. 1998) such as: Kir2, Kir3 (Kobrinsky et al. 2000; Cho et al. 2001; Lei et al. 2001; Meyer et al. 2001), KATP (Hilgemann & Ball, 1996; Shyng et al. 2000), KCNQ2/3 (Suh & Hille, 2002; Zhang et al. 2003), and TRP channels (Runnels et al. 2002; Prescott & Julius, 2003; Hardie, 2003).
, 百拇医药
Here, we investigated the signal transduction mechanism of Kir3 inhibition produced by SP. The main experiments have been done using the HEK293 heterologous system. We constructed mutant Kir3 and Kir2 channels in which the strength of channel binding to PIP2 was altered. The time courses of SP-induced inhibition of these mutant channels, in comparison with the wild type channels, indicate that depletion of PIP2 does not explain the SP-induced inhibition of Kir3 channels. Co-immunoprecipitation experiments have revealed that Gq interacts with Kir3.2, but not with Kir2.2 or Kir2.1. These data suggest that Gq activation quickly inhibits Kir3 channels, possibly by direct binding of Gq to the channel. A preliminary account of this work has been published (Koike et al. 2003).
, http://www.100md.com
Methods
Primary culture of brain neurones
Neurones of the nucleus basalis were cultured from 3- to 4-day-old postnatal rats as previously described (Nakajima et al. 1985; Nakajima & Masuko, 1996). The rats were anaesthetized with ether, and after the rats became completely unconscious, the forebrain was removed, immediately followed by decapitation. This protocol was reviewed and approved by the Animal Care Committee of the University of Illinois at Chicago. The removed brain was sectioned, and the nucleus basalis was isolated from brain slices under a dissection microscope. The nuclei were dissociated with papain, plated on a glial feeder layer, and cultured at 37°C with 10% CO2. The culture medium was a modified minimum essential medium with Earle's salt (88%; Gibco BRL) containing heat-inactivated rat serum (2%, prepared in our laboratory) and 50 ng ml–1 of 2.5 S nerve growth factor. The medium had been kept overnight in glia cultures (conditioned medium) (Baughman et al. 1991).
, 百拇医药
Culture and transfection of HEK293 cells
Human embryonic kidney 293 (HEK293 or HEK293A) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U ml–1) and streptomycin (100 μg ml–1) at 37°C with 10% CO2. The cells were transfected by using Effectene Transfection Reagent (Qiagen, Valencia, CA, USA). Kir3.1 and Kir3.2 cDNAs were from the rat, and G1 and G2 cDNAs were of bovine origin. SP receptor cDNA was human (Takeda et al. 1991). Mutants were constructed by a mutagenesis kit (QuikChange; Stratagene; La Jolla, CA, USA). Each mutation was verified by the sequence facility at the University of Chicago. Unless otherwise stated, cells were transfected in 6 cm culture dishes with the following cDNAs subcloned into plasmid pCMV5 (Andersson et al. 1989). To study the SP-induced effect on Kir3 channels, 0.2 μg SP receptor, 0.15 μg each of Kir3.1 and Kir3.2, 0.3 μg each of G1, and G2, and 0.1 μg green fluorescent protein (pEGFP-N1; Clontech) cDNAs per dish were used. To investigate the SP effects on Kir2.1 channels, 0.2 μg SP receptor, 0.3 μg Kir2.1 (Wischmeyer et al. 1995) and 0.1 μg pEGFP-N1 cDNAs per dish were transfected. The total amount of cDNAs was kept at 1.6 μg by adjusting the quantity of the empty vector. The next day cultures were re-plated on 3.5 cm culture dishes, which have a centre well (1.2 cm in diameter) coated with rat-tail collagen (Roche Molecular Biochem., Indianapolis, IN, USA). Electrophysiological experiments were performed 2–3 days after the re-plating.
, 百拇医药
To investigate the effects of anti-PIP2 antibody on the channels, HEK293A cells were transfected as follows. For experiments on Kir3.1/Kir3.2 (or their mutants), the standard amounts of cDNAs, unless otherwise noted, were: 0.15 μg each of Kir3.1 and Kir3.2 (or their mutants), 0.3 μg each of G1 and G2, and 0.1 μg of pEGFP-N1 with the total amount of plasmid adjusted to 1.6 μg by empty vector (for each 6 cm culture dish). To investigate Kir2.1 (or its mutants), we used 0.3 μg of Kir2.1, and 0.1 μg of pEGFP-N1, again the total amount of plasmid adjusted to 1.6 μg.
, 百拇医药
Electrophysiology
The whole-cell version of patch clamp was used. The external solution contained (mM): 141 NaCl, 10 KCl, 2.4 CaCl2, 1.3 MgCl2, 11 D-glucose, 5 Hepes-NaOH, and 0.0005 tetrodotoxin (pH 7.4). The patch pipette solution contained (mM): 141 potassium gluconate, 10 NaCl, 5 Hepes-KOH, 0.5 EGTA-KOH, 0.1 CaCl2, 4 MgCl2, 3 Na2ATP and 0.2 GTPS (or 0.2 GTP) (pH 7.2). The external solutions were exchanged with a sewer pipe manifold, except for certain early experiments, where a pressure ejector was used. The half-time of the SP-induced inhibition was obtained in most cells by a curve fitting program (Origin programs) with the Boltzmann or exponential functions.
, http://www.100md.com
The binding strength of the channels to PIP2 was determined by measuring the time courses of inactivation upon sudden application of anti-PIP2 antibody to the inside-out patch (Huang et al. 1998; Zhang et al. 1999). The patch pipette contained (mM): 155 KCl, 2.4 CaCl2, 1.3 MgCl2 and 5 Hepes(NaOH), pH 7.4. The bath solutions (bathing the cytoplasmic side) were similar to those described in Huang et al. (1998), containing fluoride (F) and vanadate (V) to inhibit the lipid phosphatases. The FV solution contained (mM): 146 KCl, 5 EGTA(KOH), 10 Hepes(KOH), 5 NaF, and 0.1 Na3VO4, pH 7.2. The FV-ATP solution contained (mM): 136 KCl, 5 EGTA(KOH), 10 Hepes(KOH), 5 NaF, 0.1 Na3VO4, 3 ATP(KOH) and 2 MgCl2 (pH 7.2). Anti-PIP2 monoclonal antibody was obtained from Assay Designs, Inc (Ann Arbor, MI, USA). The antibody was purified, thanks to the help of Stephen C. Lam and Joseph M. Schober (University of Illinois at Chicago), by protein A–sepharose. IgG2b for the negative control was from Sigma-RBI.
, http://www.100md.com
Physiological experiments were conducted at a bath temperature of 24°C. The statistical values are expressed as mean ± S.E.M.
Immunoprecipitation
HEK293 cells were transfected with plasmid cDNAs of Gq and either Myc-Kir3.2, T7-Kir2.2 or Myc-Kir2.1 by Trans IT-LT1 (Panvera). The amount of cDNA used for transfection was adjusted by adding pCMV5 to 20 μg total per 10 cm dish of the cells. Transfected cells from the 10 cm dish were harvested and solubilized in 550 μl of lysis buffer on ice for 20 min. The lysis buffer consisted of: 20 mM Hepes-NaOH at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 25 mM glycerophosphate, 1 mM Na3VO4, 2 μg ml–1 aprotinin, 10 μg ml–1 leupeptin, 10 μg ml–1 pepstatin A, 0.5% Triton-X 100, 10% glycerol and 10 μM GDP. In some experiments, 30 μM AlCl3 and 5 mM NaF were added to the lysis buffer to activate Gq. The samples were centrifuged at 15 000 g for 20 min at 4°C. The supernatants were subjected to immunoprecipitation. The cell lysates (150 μl) were incubated at 4°C for 1 h with antibody (0.3–0.5 μg) and protein A–Sepharose. Immune complexes with protein A–Sepharose were washed three times with a wash buffer (20 mM Hepes-NaOH at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5% Triton-X 100 and 10 μM GDP) either with or without 30 μM AlCl3 and 5 mM NaF. Immunoprecipitated proteins were boiled in a sample buffer (50 mM Tris-HCl at pH 6.8, 1% SDS, 2% 2-mercaptoethanol, 0.008% bromophenol blue, and 8% glycerol) and resolved by SDS-PAGE. After SDS-PAGE, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with blocking buffer (50 mM Tris-HCl at pH 8.0, 2 mM CaCl2, 80 mM NaCl, 5% skim milk, 0.2% NP-40 and 0.02% NaN3), and proteins were immunoblotted with antibodies described below. The bound antibodies were visualized by the enhanced chemiluminescence detection system (Pierce), using antimouse or antirabbit Ig antibodies conjugated with horseradish peroxidase as secondary antibodies (Amersham). Rabbit polyclonal antibody which recognizes Gq/11 was purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody 9E10 against c-Myc epitope was from Covance. Mouse monoclonal antibody against T7 epitope was from Novagen.
, 百拇医药
Results
Primary cultured brain neurones
It has been shown that sympathetic neurones can be transfected by introducing cDNAs into cell nuclei by a microinjector (Ikeda, 1997). Figure 1A–C shows that our primary cultured brain neurones from the nucleus basalis can be transfected using the same microinjection method.
A–C, primary cultured neurones from the nucleus basalis transfected by intranuclear injection of cDNA for GFP, G1 and G2. The injection pressure was 30 hPa for 0.1 s. The micrographs were taken without fixation. A, fluorescence micrograph. B and C, microscopic view of a neurone with phase-contrast in B, and fluorescence optics in C. Calibration bars: 100 μm (in A) and 50 μm (in B and C). D and E, SP-induced Kir current inhibition in nucleus basalis neurones. Injection of cDNA for RGS4 reduced the rate of SP-induced Kir inhibition. Each neurone received an intranuclear injection of cDNAs for M2-muscarinic receptors (0.3 μg μl–1), RGS4 (or empty vector) (0.5 μg μl–1), and GFP (0.1 μg μl–1). Whole-cell recordings were done 30–50 h after the injection. Holding potential, –79 mV. Command voltages of 20 mV depolarization and 50 mV hyperpolarization (each 100 ms duration) were imposed once every 4 s. The current change upon the 50 mV hyperpolarization minus the Ba2+ (100 μM)-resistant current was taken as amplitude of the Kir current. First, muscarine (20 μM) was applied to activate Kir3 channel (not shown in the figures), followed by SP (0.3 μM) application. Control: SP application caused a fast inhibition of the Kir channels (in D). A neurone injected with RGS4 cDNA showed a slow inhibition by SP (in E). F, speed of the SP-induced inhibition of the Kir channel. *P < 0.05. The diameter of the neurones was: control, 29.3 ± 1.3 μm (n = 8); RGS4, 29.8 ± 1.6 μm (n = 6). These large neurones are most probably cholinergic (Nakajima et al. 1985).
, 百拇医药
In Fig. 1D–F, cultured nucleus basalis neurones were transfected, using the microinjection method, with cDNAs for M2-muscarinic receptor, RGS4 (or empty vector for control), and GFP. We then performed whole-cell recordings using pipettes containing GTPS (200 μM). About 30 s after the giga-seal was broken, we started recording the conductance. The inward rectifier conductance (measured with 50 mV hyperpolarization) began to increase, reaching a plateau in about 5 min. This increase results from activation of endogenous Gi protein caused by spontaneous exchange of GDP (bound to Gi) with GTPS; this in turn would result in the liberation of G, which opens the Kir3 channels. In the present experiments, while the conductance was increasing, we activated the M2-muscarinic receptors by muscarine (20 μM), which further accelerated the conductance increase. After reaching a plateau, the conductance often started to decrease.
, 百拇医药
Application of SP (0.3 μM) to a control neurone after the conductance reached a plateau caused a rapid reduction of the conductance (Fig. 1D). SP receptor activation causes a rapid dissociation of Gq from the G subunit; Gq then, through an unknown mechanism, inhibits the Kir3 channels. Because the patch pipette contained GTPS, the SP application activated the receptor almost maximally without complications from receptor desensitization. The GTPS-bound G and the isolated G remain separated indefinitely.
, 百拇医药
As shown in Fig. 1E and F, the rate of the SP-induced inhibition was considerably reduced in neurones in which RGS4, an inhibitor of Gq and Gi (Hepler et al. 1997), had been additionally expressed. As there are two types of Kir channels that are affected by SP in nucleus basalis neurones (Bajic et al. 2002), the interpretation is not straightforward. Nevertheless, the present result is consistent with the notion that Gq mediates the SP-induced inhibition of native Kir3 channels in brain neurones, in agreement with conclusions from heterologous systems by Leaney et al. (2001) and Lei et al. (2001).
, http://www.100md.com
An explanation is needed on the rationale for our experimental protocol in Fig. 1D–F. The rationale relies on the difference between Gq and Gi in their activation kinetics in the presence of GTPS. Shortly after the break of the patch, the conductance begins to increase spontaneously (without receptor activation) in the presence of GTPS; this probably reflects spontaneous dissociation of Gi from G caused by an exchange of GDP with GTPS. At the peak of the conductance, activation of Gq by SP application produces a large inhibition, suggesting that the spontaneous activation of Gq by GTPS is much slower than the spontaneous activation of Gi by GTPS. Biochemical data using an in vitro preparation indicate that in order to measure the spontaneous replacement of GDP by GTPS, a 100 times higher GTPS concentration is needed for Gq than for Gi (Linder et al. 1990; Hepler et al. 1993); this means that the rate of the replacement, at the same GTPS concentration, would be much slower for Gq than for Gi.
, http://www.100md.com
RGS4 inhibits not only Gq, but also Gi. Therefore, possible inhibition of Gi by RGS4 could affect the initial spontaneous conductance increase in the presence of GTPS. Our data, however, showed that the conductance increase, which was caused by the combination of spontaneous and muscarine-induced activation, was almost the same in control and in the RGS4-transfected cells. The conductance reached a level of 203 ± 36% during the initial 254 ± 16 s in control cells (n = 8), and to a level of 220 ± 30% during the initial 304 ± 31 s in the RGS4-transfected cells (n = 8). These results indicate that the initial conductance increase was not much affected by the presence of RGS4. This is expected because RGS inhibits the G subunit only after it is split from the G, and the conductance increase of Kir3 is mediated by G not by G. By the time SP was applied, almost all the Gi had probably been fully activated in part by the spontaneous GDP–GTPS exchange, and in part by the muscarine application. Therefore, it is likely that the SP-induced conductance inhibition is caused by the Gq subunit, not by Gi, for both control and the RGS-transfected cells.
, http://www.100md.com
HEK293 cells: role of Gq
From this point on, all data were obtained from HEK293 cells. To examine the mechanism of the SP-induced Kir3 inhibition, we used HEK293 cells expressing the SP receptor, Kir3.1, Kir3.2, G1, G2 and GFP. In these cells, the Kir3.1/Kir3.2 channels were active from the beginning because of the co-transfection of G1 and G2. Application of SP to these cells produced a fairly quick inhibition (Fig. 2A). The half-time of the inhibition (T0.5) was 11 s (Fig. 2C), which is about the same as that of the SP-induced inhibition of native Kir3 channels in locus coeruleus neurones (12 s; Velimirovic et al. 1995). The N-terminus construct of G protein-coupled receptor kinase 2 (GRK2-nt) specifically interacts with the active form of Gq, but not with Gi (Carman et al. 1999). When HEK293 cells were transfected with GRK2-nt cDNA, in addition to the control set of cDNAs, the rate of the Kir3 inhibition (k0.5) became considerably slower (Fig. 2B and C), suggesting that SP inhibited the Kir3.1/Kir3.2 channels through Gq.
, 百拇医药
A, HEK293 cells transfected with SP receptor, wild type Kir3.1/Kir3.2 and G12. Application of SP (0.5 μM) inhibited the Kir3 current rapidly. Whole-cell current with the pipette solution containing GTPS (200 μM). Holding potential, –79 mV. Command voltages of 20 mV depolarization and 50 mV hyperpolarization (each 100 ms duration) were imposed once every 4 s. The amplitude of current change upon the 50 mV hyperpolarization was taken as the magnitude of the Kir current. The above protocol was used as a standard procedure in the following experiments unless otherwise stated. See Kawano et al. (1999) and Zhao et al. (2003) for the current–voltage relation recorded from HEK293 cells using a similar transfection protocol. B, rate of the inhibition (k0.5) (see text) was reduced by transfecting the cells with GRK2-nt (Carman et al. 1999), which interacts with the active form of Gq. C, comparison of the rate of SP-induced inhibition between the control (Kir3.1/Kir3.2) and the test (Kir3.1/Kir3.2 with GRK2-nt). *P = 0.0101. HEK293 cells were transfected with the standard protocol plus either 0.8 μg of empty vector (control) or GRK2-nt cDNA.
, 百拇医药
In Fig. 2, as well as in the following figures, we expressed the rate of the inhibition as the reciprocal of half-time, k0.5 = (T0.5)–1. In the present experiments, k0.5 was a better behaved variable than half-time. If half-time was used, occasionally one cell with an unusually large half-time resulted in a large arithmetic mean, overshadowing the behaviour of other cells.
Kir3 mutants
The binding strength of a channel to PIP2 is an important determinant of the channel activity (Hilgemann & Ball, 1996; Huang et al. 1998; Zhang et al. 1999). A convenient way to assess the strength of interaction is to apply anti-PIP2 antibody suddenly to the inside of the membrane and measure the rate of the conductance decrease (Huang et al. 1998). The rate is related to the ‘binding strength of the channel to PIP2’: the stronger the binding, the slower the speed of channel inactivation by the antibody (i.e. smaller value of k0.5). Affinity of the channel (reciprocal of the dissociation constant) to PIP2 can be measured by a dose–response relationship between water-soluble PIP2 and channel activity (Rohacs et al. 2002; Lopes et al. 2002; Zhang et al. 2003). Under most conditions, the two variables (the binding strength and the affinity to PIP2) would parallel each other. In fact, Logothetis' group showed that the binding strength of the channel to PIP2 measured by the antibody in various Kir2.1 mutants parallels the channel activity (Lopes et al. 2002), which in turn would represent the ‘affinity’ of the channel.
, 百拇医药
According to Zhang et al. (1999), the binding strength to PIP2 (as measured by anti-PIP2 antibody) is higher in mutant Kir3.4(I229L) than in the wild type Kir3.4. We therefore constructed Kir3.1 and Kir3.2 mutants at the residues corresponding to Kir3.4(I229L) (namely, Kir3.1(M223L) and Kir3.2(I234L)). HEK293 cells were then transfected with these mutant Kir3s. The strength of the binding of the mutant Kir3 channel to PIP2 was then examined by applying a monoclonal antibody against PIP2 to inside-out patches. As shown in Fig. 3A–C, the purified monoclonal antibody against PIP2 (50 nM) eliminated the activity of the wild type Kir3.1/Kir3.2 with a half-time of 29 s, while the same antibody treatment hardly affected the activity of Kir3.1(M223L)/Kir3.2(I234L) over several minutes (see also Fig. 3D). As a negative control, we used purified IgG2b (120 nM), which did not change the Kir3 activity (4 cells). These data, in agreement with Zhang et al. (1999), indicate that Kir3.1(M223L)/Kir3.2(I234L) has a much stronger binding to PIP2 than wild type Kir3.1/Kir3.2.
, http://www.100md.com
A and B, an inside-out patch was made from a HEK293A cell transfected with wild type Kir3.1/Kir3.2 plus G12 or Kir3.1(M223L)/Kir3.2(I234L) plus G12. Holding potential, –90 mV. The patch was first perfused with FV solution (Methods), followed by the Mg2+-ATP-containing FV solution (ATP), and finally by anti-PIP2 antibody-containing FV solution (Ab). C and D, the antibody produced a very slow inhibition in mutant Kir3.1(M223L)/Kir3.2(I234L). In C, purified monoclonal antibody was used at 50 nM. **P < 0.01. In D, a non-purified monoclonal antibody was used at 1 : 50 dilution. P = 0.06. E–G, SP-induced inhibition in whole-cell recording with pipettes containing GTPS. H–J, SP-induced inhibition with patch-pipettes containing 0.2 mM GTP. The recovery from the SP effect was measured 180 s after the peak of the SP effect: 15.5 ± 3.3% (n = 6) for the wild type and 17.8 ± 3.3% (n = 6) for the mutant. The difference is not significant.
, 百拇医药
Interestingly, the rate of SP-induced inhibition of Kir3.1(M223L)/Kir3.2(I234L) channels was essentially the same as that of the wild type Kir3.1/Kir3.2 (Fig. 3E–G). If anything, the rate of SP-induced inhibition for the mutant channels was slightly higher, in contrast to what the antibody data would suggest (Fig. 3G). This result does not agree with the idea that SP-induced Kir3 inhibition originates from depletion of PIP2 (Kobrinsky et al. 2000). If the SP-induced inhibition were caused by PIP2 depletion only, then Kir3.1(M223L)/Kir3.2(I234L), which binds strongly to PIP2, would have hardly been inhibited by the SP application.
, 百拇医药
The above experiments were all performed in the presence of internal GTPS. In contrast, the experiments in Fig. 3H–J were performed under more physiological conditions using a patch pipette solution containing GTP instead of GTPS. The result shows that the rate of SP-induced inhibition (k0.5) of the mutant channel Kir3.1(M223L)/Kir3.2(I234L) was again almost the same as that of the wild type Kir3.1/Kir3.2 (for the experiments with GTP, T0.5 was 14 s in control and 12 s in the mutant). We also measured the magnitude of conductance reduction by SP application in experiments with GTP-containing solution. The reduction was essentially the same between the wild type and the mutant; namely, for the wild type it was 51.2 ± 5.7% (n = 7) and for the mutant 49.2 ± 6.1% (n = 6). In conclusion, the results of these experiments show that the SP-induced Kir3 inhibition cannot be attributed to depletion of PIP2.
, 百拇医药
Kir2.1 (R218Q)
Kir2.1(R218Q) is a mutant with weak binding to PIP2 (Zhang et al. 1999). The channel activity of this mutant Kir2.1 therefore is readily inhibited by a small decline of the PIP2 level (Kobrinsky et al. 2000). We constructed Kir2.1(R218Q) and examined its responses to anti-PIP2 antibody and to SP application. In agreement with the result by Zhang et al. (1999), the inactivation rate by the antibody was faster in Kir2.1(R218Q) than that of the wild type Kir3.1/Kir3.2, indicating that the mutant Kir2.1(R218Q) binds to PIP2 less strongly than does the wild type Kir3.1/Kir3.2 (Fig. 4A and B).
, 百拇医药
A and B, anti-PIP2 antibody-induced inactivation of the currents recorded from inside-out patches. Holding potential, –90 mV. After activating the channel with Mg2+-ATP in FV solution, anti-PIP2 antibody (Ab) in FV solution was applied (time zero). Each symbol on the curves represents normalized mean current amplitude (n = 6). A non-purified monoclonal antibody at 1 : 200 dilution was used. *P < 0.05. C–E, SP-induced inhibition of the whole-cell current, with the pipette containing 0.2 mM GTPS. Protocol of DNA transfection was: 0.5 μg SP receptor, 0.2 μg each of Kir3.1, Kir3.2, G1 and G2 (or 1.0 μg of Kir2.1(R218Q)), and 0.1 μg of GFP (with total adjusted to 1.6 μg by pCMV5). We have sometimes noticed that SP application produced a small inward current, followed by the conductance decrease (inhibition of Kir channels) in HEK293 cells. Analysis of these transient inward currents in primary cultured neurones indicated that the transient inward current was caused by activation of non-selective cation channels (Farkas et al. 1996). We further noticed that an increase in the external divalent cation concentration tended to inhibit the non-selective cation channels. We therefore used a high Ca2+ concentration (20 mM) external solution in the hope of diminishing possible activation of non-selective cation channels (both for Kir3.1/Kir3.2 and for Kir2.1(R218Q)) (C–E), but the transient inward current still occurred. E, summary; **P < 0.01. We repeated the same experiment either with the standard conditions (Methods) or with a slight modification (internal Mg2+-ATP was 2 mM, instead of 3 mM). The overall results were: mean of k0.5 for wild type Kir3.1/Kir3.2 was 0.103 s–1 (n = 18) and that for Kir2.1(R218Q) was 0.0228 s–1 (n = 10).
, http://www.100md.com
The rates of SP-induced inhibition of these channels, however, contradicted those from PIP2 reduction. If the SP-induced inhibition was caused by PIP2 depletion, the inhibition of Kir2.1(R218Q) should occur faster than that of the wild type Kir3.1/Kir3.2. On the contrary, the time course of SP-induced inhibition of Kir2.1(R218Q) was considerably slower than that of the wild type Kir3.1/Kir3.2 (Fig. 4C–E) (P < 0.001). The result indicates that SP-induced Kir3 inhibition arises from a mechanism that is independent of PIP2 depletion.
, 百拇医药
Kir2.1
According to Zhang et al. (1999), the rank order of the binding strength of various Kir2.1 s to PIP2, as measured by the antibody, is: wild type Kir2.1 > Kir2.1(R228Q) > Kir2.1(R218Q) (Zhang et al. 1999). We examined the SP-induced inhibition of Kir2.1 channels with whole-cell recordings using patch pipettes containing GTPS. In wild type Kir2.1, we usually did not observe a SP-induced inhibition; only in 1 out of 5 cells did SP application result in a very slow inhibition. The mutant Kir2.1(R228Q) responded to SP more readily: 4 out of 7 were clearly inhibited by SP with an approximate value of k0.5 between 0.03 and 0.005 s–1. As mentioned in the previous section, the mutant Kir2.1(R218Q), which binds PIP2 weakly, showed a SP-induced inhibition with a mean k0.5 of 0.0178 s–1.
, 百拇医药
These data, although not amenable to statistical analysis, suggest that the rank order of difficulty of SP-induced channel inhibition is: Kir2.1 > Kir2.1(R228Q) > Kir2.1(R218Q). This rank order is compatible with the known order of binding strength for PIP2. Thus, as long as we consider only the responses of Kir2.1 and its mutants, the data are compatible with the idea that the SP-induced response originates from PIP2 depletion.
During this set of experiments, we also recorded the SP-induced inhibition of wild type Kir3.1/Kir3.2 (in the presence of G). The rate for Kir3.1/Kir3.2 was fast (k0.5 = 0.087 ± 0.011 s–1; n = 8) and was essentially the same as that for Kir3.1/Kir3.2 in other sets of experiments. Thus, the SP-induced inhibition rate for wild type Kir2.1 as well as that for Kir2.1(R228Q) was much slower than that of Kir3.1/Kir3.2 (in the presence of G).
, 百拇医药
The conclusion of this section is that (a) if we focus on the behaviour of Kir2.1 and its mutants, the SP-induced inhibition is compatible with the notion that inhibition originates from the depletion of PIP2, and (b) the SP-induced inhibition of Kir3.1/Kir3.2 is unique and far faster than expected from the depletion of PIP2.
Role of protein kinase C
The role of protein kinase C (PKC) in the inhibition of Kir3 channels has been a controversial subject. Using the Xenopus oocyte expression system, Sharon et al. (1997) showed that staurosporine (non-specific protein kinase inhibitor) or bisindolylmaleimide (PKC inhibitor affecting the catalytic domain) almost completely blocks the transmitter-induced inhibition of basal Kir3 current. Mao et al. (2004) showed that treatment of oocytes with calphostin C (PKC inhibitor acting on the regulatory domain) or chelerythrine (PKC inhibitor acting on the catalytic domain) completely blocks the SP-induced Kir3 inhibition (see also Hill & Peralta, 2001).
, 百拇医药
However, results obtained using mammalian host cells (HEK293) are different. Leaney et al. (2001) report that treatment with bisindolylmaleimide I or Ro-31-8220 (3 μM for at least 5 min) reduces the muscarine-induced inhibition of the basal activity of Kir3.1/Kir3.2 channels by 40–60%. Lei et al. (2001) find that application of phorbol ester produces little effect on activated (not basal) Kir3.1/Kir3.2 currents in HEK293 cells. Nor does the application of bisindolylmaleimide I (5 μM; > 30 min) have any effect on the TRH-induced inhibition of activated Kir3 channels.
, 百拇医药
We tested the effect of bisindolylmaleimide I (3 μM, for 10–30 min) in the presence of internal GTPS. As shown in Fig. 5C and D, application of the inhibitor did not change the rate (k0.5) of the SP-induced inhibition of activated Kir3.1/Kir3.2 channels (Fig. 5C). The magnitude of the SP-induced inhibition became slightly less through the application of bisindolylmaleimide I (but statistically not significant; Fig. 5D). These results agree with those of Lei et al. (2001). The most conspicuous effect produced by bisindolylmaleimide I was that the Kir3.1/Kir3.2 current itself was reduced considerably (Fig. 5E). It is interesting to note that despite the reduced conductance of Kir3.1/3.2 caused by the PKC inhibitor, the mechanism to produce the quick inhibition by SP seems mostly intact. This is in sharp contrast to the effect of the Gq inhibitor GRK-nt (Fig. 2), in which the speed of the SP-induced inhibition became slower.
, 百拇医药
HEK293 cells were transfected with cDNAs for SP receptor, Kir3.1, Kir3.2, G1 and G2 (see Methods). A, a control cell with 0.1% DMSO in the external solution. See Fig. 2 legend for the sequence of the command voltage pulses. Application of SP (1 μM) inhibited Kir3.1/Kir3.2 current. The current amplitude upon 50 mV hyperpolarization was taken as representing Kir3.1/Kir3.2 current. B, BIS-treated cells. C, the rate (k0.5) of the SP-induced inhibition was not affected by the BIS treatment (3 μM, for 11–36 min). D, the magnitude of the SP-induced Kir3.1/Kir3.2 current inhibition relative to control current. E, the BIS treatment considerably reduced the Kir3.1/Kir3.2 current (before the SP application). Ordinate: current amplitude produced by a 50 mV hyperpolarization divided by the input capacitance. *P < 0.05.
, http://www.100md.com
We can summarize the above results from different investigators as follows: when expressed in mammalian cells, the role of PKC in the receptor-mediated inhibition of strongly activated Kir3 channels seems to be small, whereas PKC does play a moderate role in inhibiting the basal activity of Kir3 channels. On the contrary, PKC does play a substantial role in the inhibition of Kir3 channels expressed in Xenopus oocytes.
Gq physically interacts with Kir3 but not with Kir2
, http://www.100md.com
The above results suggest that the SP effect on Kir3.1/Kir3.2 is faster than and of different origin from the SP effect on Kir2. There must be another quicker signalling route for Kir3.1/Kir3.2 inhibition. To elucidate the mechanism of this signalling pathway, we examined the interaction of Gq with Kir3 or with Kir2 by co-immunoprecipitation. Previously, Simen et al. (2001) reported that Gq and a type of Ca2+ channel physically interact with each other.
We found that Gq co-precipitated with Kir3.2 (Fig. 6A), whereas Gq did not co-precipitate with either Kir2.2 (Fig. 6B) or with Kir2.1 (Fig. 6C). Both the transition state form (GDP-AlF4–-bound form) and the GDP-bound form of Gq were co-precipitated with Kir3.2 (Fig. 6A), but not with Kir2.2 or Kir2.1 (Fig. 6B and C).
, http://www.100md.com
A, Gq associates with Myc-Kir3.2. (IP: Myc) Anti-Myc antibody co-precipitated Gq from cells expressing both Gq and Myc-Kir3.2 (lane 5 and lane 6). (IP: Gq) Anti-Gq/11 antibody co-precipitated Myc-Kir3.2 from cells expressing both Gq and Myc-Kir3.2. Both inactive (without AlF4–) and active (with AlF4–) forms of Gq were co-immunoprecipitated with Kir3.2. Experiments were performed using two different protocols: (i) wild type Gq with AlF4– (lane 6) or without AlF4– (lane 5), and (ii) constitutively active Gq (R183C) (without AlF4–). The results were the same for the two protocols. A representative result from 4 experiments (2 from each one of the protocols) is shown. B, Gq does not co-precipitate with Kir2.2. (IP: T7) Anti-T7 antibody did not co-precipitate Gq from cells expressing both Gq and T7-Kir2.2, while it co-precipitated T7-Kir2.2 from cells expressing T7-Kir2.2 alone (lane 4) or together with Gq (lane 5 and lane 6, top panel). (IP: Gq) Anti-Gq/11 antibody did not co-precipitate T7-Kir2.2 from cells expressing both Gq and T7-Kir2.2 (lane 5 and lane 6), whereas it co-precipitated Gq from cells expressing Gq alone (lane 2 and lane 3) and Gq with T7-Kir2.2 (lane 5 and lane 6). A representative result from 3 experiments is shown; each includes data with and without GDP-AlF4–. In addition two experimental runs were performed using Gq(R183C). These two different protocols produced the same result. C, Gq does not co-precipitate with Kir2.1. A representative result from 5 experiments is shown. * in A and C indicates IgG heavy chains of anti-Myc antibody.
, 百拇医药
Discussion
The present results show that the speed of SP-induced Kir3 inhibition was much faster than predicted from the assumption that Kir3 inhibition originates from PIP2 reduction. Although the binding strength of Kir3 to PIP2 was increased considerably (90 times) by mutation (Kir3.1(M223L)/Kir3.2(I234L)) (Fig. 3), the rate of SP-induced inhibition of the mutant channel was unchanged. As for experiments on Kir2.1(R218Q) (Fig. 4), the antibody-induced inhibition rate of Kir3.1/Kir3.2 was 2.5 times slower than that of Kir2.1(R218Q) (Fig. 4B), whereas the SP-induced inhibition of Kir3.1/Kir3.2 was 7.5 times faster than that of Kir2.1(R218Q). Hence, the SP-induced Kir3 inhibition is 19 times faster than expected from PIP2 binding characteristics. This kind of comparison, assuming a very strong affinity of the antibody to PIP2 and linear relations over large ranges, would have a considerable quantitative inaccuracy. Nonetheless, it indicates that the rapidity of the SP-induced Kir3 inhibition is by no means trivial. Thus, PIP2 reduction cannot account for the direct cause for the SP-induced inhibition of Kir3.1/Kir3.2 channels. There must be a mechanism in Kir3 channels which renders them capable of responding so rapidly to the transmitter. It is unlikely that this mechanism involves PKC. The experiments by Lei et al. (2001) and ours (Fig. 5) on the PKC inhibitor bisindolylmaleimide I indicate that, unlike the results obtained using Xenopus oocyts (Sharon et al. 1997; Hill & Peralta, 2001; Mao et al. 2004), PKC seems to play a relatively small role in the SP-induced inhibition of activated Kir3 channels expressed in mammalian host cells.
, 百拇医药
Recently, a GFP-tagged PH domain of PLC has been used as an optical probe to observe PIP2 hydrolysis (Runnels et al. 2002; Rohacs et al. 2002; Zhang et al. 2003; Suh et al. 2004). The data show that PIP2 hydrolysis by receptor activation occurs as fast as the rate of channel inhibition. These data support the notion (Suh & Hille, 2002; Zhang et al. 2003; Suh et al. 2004) that transmitter-induced inhibition of KCNQ2/3 is primarily caused by depletion of PIP2. Is our conclusion compatible with these findings A crucial factor to consider is the large variation in the PIP2 affinity among different types of channels. The PIP2 affinity of KCNQ2/3 is much less than that of Kir2.1, with an EC50 of 90 μM for KCNQ2/3, and 5 μM for Kir2.1 (EC50 is measured with a water-soluble diC8-PIP2; Rohacs et al. 2002; Zhang et al. 2003). This means that KCNQ2/3 could be inhibited by a small decline of PIP2, while Kir3 (in the presence of G) or Kir2, with its inherently high PIP2 affinity (Huang et al. 1998), may remain active even with a considerably reduced level of PIP2. Therefore, the conclusion drawn from KCNQ studies does not necessarily contradict our conclusion from Kir3 studies. Importantly, the optical experiments do not describe the relative amount of PIP2 remaining inside the membrane after transmitter application.
, 百拇医药
Previously, Huang et al. (1995) and Peleg et al. (2002) showed, using the glutathione S-transferase (GST) pull down assay, that Gi and/or G interact with the Kir3 N-terminus. Furthermore, Dascal's group (Schreibmayer et al. 1996; Peleg et al. 2002) has shown that Gi inhibits the basal Kir3 activity independent of its ability to sequester G. Thus, Gq and Gi, despite mediating opposite gross effects on Kir3 channel activity, may actually share certain signalling capabilities. The question is to explain these opposite effects on channel activity despite the fact that both Gi and Gq seem to be capable of inhibiting channel activity. Furthermore, channel-activating G is liberated regardless of the G protein system under stimulation. The functional differences may arise simply due to the quantitative strengths of the involved G protein subunits. The capability of Gq to inhibit Kir3 is strong enough to nullify the activating effect of G (Velimirovic et al. 1995), whereas channel inhibition by Gi is so weak as to be easily overcome by G.
, http://www.100md.com
We have presented two novel findings from the present research: (a) a unique mechanism must exist between Gq and Kir3.1/Kir3.2 to produce inhibition of Kir3. This mechanism does not exist between Gq and Kir2.1; (b) there is a physical interaction between Gq and Kir3.2, but not between Gq and Kir2.1 or Kir2.2. Furthermore, the literature shows that there is some similarity between Gi and Gq in their capacity to inhibit Kir3 activity (Peleg et al. 2002) and to physically interact with Kir3 (Huang et al. 1995; Peleg et al. 2002). A simple hypothetical mechanism that explains all these facts might be a signalling pathway that includes a protein–protein interaction between Gq and Kir3, resulting in closure of the channel gate.
, 百拇医药
Footnotes
M. Koike-Tani and J. M. Collins contributed equally to this work.
References
Adams PR, Brown DA & Jones SW (1983). Substance P inhibits the M-current in bullfrog sympathetic neurones. Br J Pharmacol 79, 330–333.
Andersson S, Davis DL, Dahlback H, Jornvall H & Russell DW (1989). Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem 264, 8222–8229.
, 百拇医药
Bajic D, Koike M, Albsoul-Younes AM, Nakajima S & Nakajima Y (2002). Two different inward rectifier K+ channels are effectors for transmitter-induced slow excitation in brain neurons. Proc Natl Acad Sci U S A 99, 14494–14499.
Baughman RW, Huettner JE, Jones KA & Khan AA (1991). Cell culture of neocortex and basal forebrain from postnatal rats. In Culturing Nerve Cell, ed. Baker G & Goslin K, pp. 227–249. MIT Press, Cambridge, Massachusetts.
, 百拇医药
Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedegaertner PB, Gilman AG, Benovic JL & Kozasa T (1999). Selective regulation of Galpha (q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. J Biol Chem 274, 34483–34492.
Chang MM & Leeman SE (1970). Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J Biol Chem 245, 4784–4790.
Cho H, Nam GB, Lee SH, Earm YE & Ho WK (2001). Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in alpha (1) -adrenergic pathway via the modulation of acetylcholine-activated K+ channels in mouse atrial myocytes. J Biol Chem 276, 159–164.
, http://www.100md.com
Dam T-V & Quirion R (1994). Comparative distribution of receptor types in the mammalian brain. In The Tachykinin Receptors, ed. Buck SH, pp. 101–123. Humana Press, Totowa, New Jersey.
Dascal N et al. (1993). Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc Natl Acad Sci U S A 90, 10235–10239.
Farkas RH, Chien PY, Nakajima S & Nakajima Y (1996). Properties of a slow nonselective cation conductance modulated by neurotensin and other neurotransmitters in midbrain dopaminergic neurons. J Neurophysiol 76, 1968–1981.
, http://www.100md.com
Farkas RH, Chien PY, Nakajima S & Nakajima Y (1997). Neurotensin and dopamine D2 activation oppositely regulate the same K+ conductance in rat midbrain dopaminergic neurons. Neurosci Lett 231, 21–24.
Hardie RC (2003). Regulation of TRP channels via lipid second messengers. Annu Rev Physiol 65, 735–759.
Hepler JR, Berman DM, Gilman AG & Kozasa T (1997). RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. Proc Natl Acad Sci U S A 94, 428–432.
, 百拇医药
Hepler JR, Kozasa T, Smrcka AV, Simon MI, Rhee SG, Sternweis PC & Gilman AG (1993). Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits. J Biol Chem 268, 14367–14375.
Hilgemann DW & Ball R (1996). Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2. Science 273, 956–959.
Hill JJ & Peralta EG (2001). Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor. J Biol Chem 276, 5505–5510.
, http://www.100md.com
Huang CL, Feng S & Hilgemann DW (1998). Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G beta gamma. Nature 391, 803–806.
Huang CL, Slesinger PA, Casey PJ, Jan YN & Jan LY (1995). Evidence that direct binding of G beta gamma to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation. Neuron 15, 1133–1143.
Ikeda SR (1997). Heterologous expression of receptors and signaling proteins in adult mammalian sympathetic neurons by microinjection. Meth Mol Biol 83, 191–202.
, 百拇医药
Kawano T, Chen L, Watanabe SY, Yamauchi J, Kaziro Y, Nakajima Y, Nakajima S & Itoh H (1999). Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K+ channels. FEBS Lett 463, 355–359.
Kobrinsky E, Mirshahi T, Zhang H, Jin T & Logothetis DE (2000). Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat Cell Biol 2, 507–514.
Koike M, Collins J, Kawano T, Zhao P, Zhao Q, Kozasa T, Nakajima S & Nakajima Y (2003). Signal transduction of GIRK inhibition by substance P. Biophys J 84, 226a.
, http://www.100md.com
Kubo Y, Reuveny E, Slesinger PA, Jan YN & Jan LY (1993). Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364, 802–806.
Leaney JL, Dekker LV & Tinker A (2001). Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca2+ -independent protein kinase C. J Physiol 534, 367–379.
Lei Q, Talley EM & Bayliss DA (2001). Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G (alpha) q family subunits, phospholipase C, and a readily diffusible messenger. J Biol Chem 276, 16720–16730.
, http://www.100md.com
Linder ME, Ewald DA, Miller RJ & Gilman AG (1990). Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli. J Biol Chem 265, 8243–8251.
Logothetis DE, Kurachi Y, Galper J, Neer EJ & Clapham DE (1987). The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325, 321–326.
Lopes CM, Zhang H, Rohacs T, Jin T, Yang J & Logothetis DE (2002). Alterations in conserved Kir channel–PIP2 interactions underlie channelopathies. Neuron 34, 933–944.
, http://www.100md.com
Mao J, Wang X, Chen F, Wang R, Rojas A, Shi Y, Piao H & Jiang C (2004). Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. Proc Natl Acad Sci U S A 101, 1087–1092.
Meyer T, Wellner-Kienitz MC, Biewald A, Bender K, Eickel A & Pott L (2001). Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes. J Biol Chem 276, 5650–5658.
, 百拇医药
Nakajima Y & Masuko S (1996). A technique for culturing brain nuclei from postnatal rats. Neurosci Res 26, 195–203.
Nakajima Y, Nakajima S, Obata K, Carlson CG & Yamaguchi K (1985). Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei. Proc Natl Acad Sci U S A 82, 6325–6329.
Nowak LM & Macdonald RL (1982). Substance P: ionic basis for depolarizing responses of mouse spinal cord neurons in cell culture. J Neurosci 2, 1119–1128.
, http://www.100md.com
Otsuka M, Konishi S & Takahashi T (1972). A further study of the motoneurons-depolarizing peptide extracted from dorsal roots of bovine spinal nerves. Proc Jpn Acad 48, 747–752.
Peleg S, Varon D, Ivanina T, Dessauer CW & Dascal N (2002). G(alpha)(i) controls the gating of the G protein-activated K+ channel, GIRK. Neuron 33, 87–99.
Prescott ED & Julius D (2003). A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity. Science 300, 1284–1288.
, http://www.100md.com
Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez-Lluhi JA, Lefkowitz RJ, Bourne HR, Jan YN & Jan LY (1994). Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. Nature 370, 143–146.
Rohacs T, Lopes C, Mirshahi T, Jin T, Zhang H & Logothetis DE (2002). Assaying phosphatidylinositol bisphosphate regulation of potassium channels. Meth Enzymol 345, 71–92.
Runnels LW, Yue L & Clapham DE (2002). The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat Cell Biol 4, 329–336.
, 百拇医药
Schreibmayer W, Dessauer CW, Vorobiov D, Gilman AG, Lester HA, Davidson N & Dascal N (1996). Inhibition of an inwardly rectifying K+ channel by G-protein alpha-subunits. Nature 380, 624–627.
Sharon D, Vorobiov D & Dascal N (1997). Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes. J General Physiol 109, 477–490.
Shyng SL, Cukras CA, Harwood J & Nichols CG (2000). Structural determinants of PIP (2) regulation of inward rectifier KATP channels. J General Physiol 116, 599–608.
, http://www.100md.com
Simen AA, Lee CC, Simen BB, Bindokas VP & Miller RJ (2001). The C terminus of the Ca channel alpha1B subunit mediates selective inhibition by G-protein-coupled receptors. J Neurosci 21, 7587–7597.
Stanfield PR, Nakajima S & Nakajima Y (2002). Constitutively active and G-protein coupled inward rectifier K+ channels: Kir2.0 and Kir3.0. Rev Physiol Biochem Pharmacol 145, 47–179.
Stanfield PR, Nakajima Y & Yamaguchi K (1985). Substance P raises neuronal membrane excitability by reducing inward rectification. Nature 315, 498–501.
, 百拇医药
Suh BC & Hille B (2002). Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35, 507–520.
Suh BC, Horowitz LF, Hirdes W, Mackie K & Hille B (2004). Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq. J General Physiol 123, 663–683.
Takeda Y, Chou KB, Takeda J, Sachais BS & Krause JE (1991). Molecular cloning, structural characterization and functional expression of the human substance P receptor. Biochem Biophys Res Commun 179, 1232–1240.
, http://www.100md.com
Vanner S, Evans RJ, Matsumoto SG & Surprenant A (1993). Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia. J Neurophysiol 69, 1632–1644.
Velimirovic BM, Koyano K, Nakajima S & Nakajima Y (1995). Opposing mechanisms of regulation of a G-protein-coupled inward rectifier K+ channel in rat brain neurons. Proc Natl Acad Sci U S A 92, 1590–1594.
Wischmeyer E, Lentes KU & Karschin A (1995). Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line. Pflugers Arch 429, 809–819.
, 百拇医药
Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T & Logothetis DE (2003). PIP2 activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37, 963–975.
Zhang H, He C, Yan X, Mirshahi T & Logothetis DE (1999). Activation of inwardly rectifying K+ channels by distinct PtdIns (4,5),P2 interactions. Nat Cell Biol 1, 183–188.
Zhao Q, Kawano T, Nakata H, Nakajima Y, Nakajima S & Kozasa T (2003). Interaction of G protein beta subunit with inward rectifier K+ channel Kir3. Mol Pharmacol 64, 1085–1091., 百拇医药(Maki Koike-Tani, John M C)