HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplementary Material All Versions of this Article: 555/3/643    most recent jphysiol.2003.056101v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Finley, M. Articles by Slesinger, P. A. Search for Related Content PubMed PubMed Citation Articles by Finley, M. Articles by Slesinger, P. A.

The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

	Abstract

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
The activity of G protein-activated inwardly rectifying K⁺ channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G_ß subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying G_ß activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G_ß binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2_L344E and GIRK2_G347H, that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of G_ß subunits. Combining the two mutations (GIRK2_EH) led to a more severe reduction in carbachol-activated and G_ß-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2_L344E and GIRK2_EH also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G_ß-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G_ß activation. In vitro G_ß binding assays revealed an 60% decrease in G_ß binding to the C-terminal domain of GIRK2_L344E but no statistical change with GIRK2_EH or GIRK2_G347H, though both mutants exhibited G_ß-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G_ß activation of GIRK2 channels. Based on the 1.8 Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the ßL–ßM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G_ß alters the interaction between the ßL–ßM loop and the N-terminal domain.

(Received 29 September 2003; accepted after revision 5 January 2004; first published online 14 January 2004)
Corresponding author P. A. Slesinger: Peptide Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. Email: slesinger{at}salk.edu

	Introduction

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
Many inhibitory neurotransmitters exert their actions by stimulating pertussis toxin (PTX)-sensitive G protein-coupled neurotransmitter receptors (GPCR), which in turn open G protein-activated inwardly rectifying K⁺ channels (GIRK or Kir3) (Hille, 1992). K⁺ ions exit the cell when GIRK channels open, thereby hyperpolarizing the cell's membrane potential and making it more difficult to elicit an action potential. The loss of GIRK channels leads to hyperexcitability and seizures in the brain (Signorini et al. 1997; Slesinger et al. 1997), cardiac abnormalities (Wickman et al. 1998) and hyperactivity and reduced anxiety (Blednov et al. 2001). Four mammalian GIRK channel subunits (GIRK1–4) have been identified, which coassemble to form neuronal GIRK channels (Yamada et al. 1998). Coimmunoprecipitation studies using brain tissues have demonstrated that GIRK channels are composed of heteromultimers of GIRK1/2 and GIRK2/3 (Liao et al. 1996; Jelacic et al. 2000). In some areas of the brain, such as in the substantia nigra, GIRK channels may exist as GIRK2 homomultimers (Liao et al. 1996; Inanobe et al. 1999).

GIRK channels have the canonical features of the inwardly rectifying K⁺ channel family (Kir1–7), which includes cytoplasmic N- and C-terminal domains, two putative transmembrane domains (M1, M2), and a highly conserved pore–loop complex involved in ion selectivity (Doupnik et al. 1995). Of the seven distinct families of Kir channels, however, only GIRK channels are activated by G protein G_ß subunits (Logothetis et al. 1987; Reuveny et al. 1994; Wickman et al. 1994). In addition, GIRK channel activity is also regulated by G protein-independent signalling molecules. Intracellular Na⁺, MgATP and PIP₂, and extracellular ethanol have all been reported to open GIRK channels in the absence of functional G proteins (Ho & Murrell-Lagnado, 1999; Kobayashi et al. 1999; Lewohl et al. 1999; Petit-Jacques et al. 1999; Zhang et al. 1999).

Since the discovery that G_ß subunits activate GIRK channels (Logothetis et al. 1987), the molecular mechanism through which G_ß subunits open the channel has remained elusive. Initially, studies using chimeras and biochemistry demonstrated that G_ß subunits bind directly to the N-terminal domain and the distal part of the C-terminal domain of GIRK1 (Huang et al. 1995; Inanobe et al. 1995; Kunkel & Peralta, 1995; Slesinger et al. 1995). Krapivinsky et al. (1998) examined the effects of synthetic peptides derived from GIRK1, GIRK4 or IRK1 on the biochemical binding of G_ß to GIRK1/4 channels and concluded that two regions within the C-terminal domain were important for G_ß binding and activation. He et al. (1999, 2002), on the other hand, examined the G_ß sensitivity of GIRK4 and IRK1 chimeras and identified specific amino acids in the N- and C-terminal domains that were important for generating either the agonist-activated or G_ß-dependent basal current (He et al. 1999, 2002). At present, there is no clear consensus on which region of the GIRK channels is essential for G_ß activation.

To identify regions important for G_ß activation, we studied chimeras of GIRK2 and a G protein-insensitive inward rectifier (IRK1). We chose GIRK2 because GIRK2, unlike GIRK1, readily forms functional homomultimers in neurones as well as in heterologous expression systems (Slesinger et al. 1996; Inanobe et al. 1999) and because the identity of amino acids involved in G_ß activation of GIRK2 is unknown. The G_ß sensitivity of chimeras was evaluated in two different expression systems: Xenopus oocytes and mammalian cells. We also examined the response of each chimeric channel to ethanol, which served as an indicator of GIRK channel gating that is G protein independent (Kobayashi et al. 1999; Lewohl et al. 1999; Zhou et al. 2001). Finally, we measured the biochemical binding of G_ß to glutathione S-transferase (GST) fusion proteins containing the C-terminal domain of the different mutant channels. We identified two amino acids in the middle of the C-terminal domain of GIRK2 that contribute to G_ß binding and activation. Some of these results have been published in the form of an abstract (Finley et al. 2003).

	Methods

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
Molecular biology

GIRK1 was in pBSK (Kubo et al. 1993b), GIRK2a cDNA was in pBTG (Lesage et al. 1994) and IRK1 was in pBSK (Kubo et al. 1993a). Chimeras were constructed by identifying transition points between GIRK2 and IRK1, using CLUSTAL alignment analysis, as illustrated in Fig. 1A. The nomenclature, I1G2_xx-xx, refers to IRK1 (I1) and the amino acid sequence in GIRK2 (G2_xx-xx) in the chimeric channel. The following point mutations in GIRK2 were tested: F338Y, T343F, L344E, G347H, F348Y and L344E/G347H (EH). All mutants were constructed using PCR and the mutation confirmed with DNA sequencing. The following chimeras were constructed but did not lead to detectable currents when expressed in oocytes: I1G2_25-190, I1G2_25-414, I1G2_25-414, I1G2_51-414, I1G2_73-414, I1G2_97-271, I1G2_1-271, I1G2_97-310, I1G2_97-335 and I1G2_97-352.

View larger version (37K): [in this window] [in a new window]   Figure 1.  The amino acid segment that differs between chimeras I1G21-335 and I1G21-352 is important for Gß activation A, schematic diagram showing design of chimeras. Examples of macroscopic currents recorded from Xenopus oocytes injected with GIRK2 (B), I1G21-335 (C), I1G21-352 (D) cRNA and either the M2 muscarinic receptor (+ M2R), or Gß1 and G2 subunits (+ Gß). GIRK2 shows both carbachol (carb)-activated and Gß-stimulated currents as well as ethanol (EtOH)-induced currents. I1G21-335 has a large basal current, but is not activated following carbachol stimulation. Currents were recorded with TEVC and perfused with 95mM KCl, 95mM KCl + 3µM carbachol (filled bar), 95mM KCl + 200mM ethanol (hatched bar) or 95mM NaCl (grey bar). The current in the presence of 95mM NaCl represents the leakage current, and was used to measure the agonist-independent basal current. Holding potential was -80 mV. E and F, bar graphs show the carbachol-induced current normalized to the basal current (E) and the agonist-independent current in the absence and presence of Gß subunits (F). *Significant difference from wild-type GIRK2 (P < 0.05). **Significant difference between basal and Gß subunits (P < 0.05). n= 5–42.

2

For expression in mammalian cells, the channel cDNA was subcloned into pcDNA3 and transfected into human embryonic kidney cells (HEK-293T). HEK-293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (10%), glutamine (2mM), penicillin (50 U ml^-1) and streptomycin (50µg ml^-1; Gibco) in a humidified 37°C incubator with 95% air–5% CO₂. Cells were plated onto 12 mm glass cover slips (Warner Instruments) coated with poly-D-lysine (20µgml^-1; Sigma) and collagen (100µgml^-1; BD Biosciences) in 24-well plates. HEK-293T cells were transiently transfected with cDNA using the calcium phosphate method. Briefly, cDNA (0.08µgml^-1) was mixed in sterile de-ionized water with 0.25M CaCl₂, then combined 1 : 1 with Hepes-buffered saline (280mMNaCl, 10mMKCl, 1.5mMNa₂HPO₄, 12mM glucose, 50mM Hepes (pH 6.9 with 1 N NaOH); 50µl of this mixture was added to each well and incubated for 16–32 h.

Electrophysiology

Macroscopic currents were recorded from oocytes with a two-electrode voltage-clamp (TEVC) amplifier (Geneclamp 500, Axon Instruments), filtered at 0.05–2kHz, digitized (0.1–2kHz) with a Digidata 1200 A/D interface (Axon Instruments) and stored on a laboratory computer. Electrodes were filled with 3M KCl and had resistances of 0.6–1 M. Oocytes were perfused continuously with an extracellular solution containing 90mM XCl (X = K⁺ or Na⁺), 2mM MgCl₂ and 10mM Hepes (pH 7.5 with 5mM XOH). The leakage current was determined using 95mM Na⁺ and subtracted directly from the current measured in 95mM K⁺. For ethanol activation, 100% ethanol was added directly to the 95mM K⁺ solution to give 200mM ethanol (EtOH density = 0.7893 g ml^-1). A small chamber (3mm x 15mm) with fast perfusion was used to change the extracellular solution and was connected to earth via a 3M KCl agarose bridge.

The whole-cell patch clamp technique (Hamill et al. 1981) was used to record macroscopic currents from HEK-293T cells. Borosilicate glass (Warner; P6165T) electrodes had resistances of 1–3 M and were coated with Sylgard to reduce capacitance. Membrane currents were recorded with an Axopatch 200B (Axon Instruments) amplifier, adjusted electronically for cell capacitance and series resistance (80–100%), filtered at 2kHz with an 8-pole Bessel filter, digitized at 5kHz with a Digidata 1200 interface (Axon Instruments) and stored on a laboratory computer. Intracellular pipette solution contained (mM) 130 KCl, 20 NaCl, 5 EGTA, 2.56 K₂ATP, 5.46 MgCl₂ and 10 Hepes (pH 7.2 with 14mM KOH). With these ion concentrations there was 140mM K⁺, 1.5mM free Mg²⁺ and 2mM Mg-ATP in the intracellular solution. Li₃-GTP (300 µM; RBI) was added fresh to the intracellular pipette solution to sustain the activation of GIRK channels. The external bath solution (20mM K⁺) contained (mM) 140 NaCl, 20 KCl, 0.5 CaCl₂, 2 MgCl₂ and 10 Hepes (pH 7.2). The osmolarity was 310–330 mosmol l^-1. Current–voltage relations were not corrected for the junction potential of 4 mV, estimated using the Junction Potential Calculator (Axon Instruments).

Biochemistry

The C-terminal domains of GIRK2 (beginning with M191) and IRK1 (beginning with V179) were subcloned into pGEX2T (Amersham Pharmacia Biotech) using BamH I and Sma I restriction sites engineered by PCR at the 5' and 3' ends of the C-terminal domains. The resulting GST-fused C-terminal domains were purified using standard procedures. G_ß binding to GST fusion proteins was measured as previously described (Huang et al. 1995) with the following modifications. Binding was performed for 45 min on ice and then each reaction sample was transferred onto a Cytosignal spin column. After three washes with 500µl PBS–0.1% lubrol, the GST-fused proteins–G_ß complexes were eluted from the columns with 15µl 2 x SDS sample buffer. Anti-G_ß antibody (SC-20: Santa Cruz) and anti-GST antibody (Amersham Pharmacia) were used for Western blot analysis. Western blots were quantified using the ‘gel’ module in Image J (NIH software). For each blot, the optical density (OD) of the G_ß band was divided by the OD for the GST band and then normalized to the GIRK2 for that experiment.

Immunocytochemistry

HEK-293T cells were cultured in DMEM containing 10% fetal bovine serum, 2.5 i.u. ml^-1 penicillin–streptomycin, and 2mM glutamine and transfected with HA-tagged constructs 24h later using the calcium phosphate method. A haemagglutinin (HA) tag was inserted into the extracellular p-loop of mutant channels by subcloning the C-terminal mutation (via the Bst EII site) into HA-GIRK2, which was kindly provided by Chen et al. (2002). Cells were fixed 20–24h after transfection by incubation in 1% paraformaldehyde for 30 min. The cells were washed two times with PBS and half were permeabilized by incubation with 0.25% Triton X-100 in PBS for 10 min. All cells were washed with PBS and incubated in blocking buffer (2% donkey serum and 2% IgG free bovine serum albumin in PBS) for 1h at room temperature. The cells were incubated in the dark in Alexa488-conjugated anti-HA antibody (Covance) in blocking buffer (1 : 400) for 2h at room temperature, washed three times with PBS and mounted onto glass coverslips with 1,4-diazabicyclo(2,2,2)octane (DABCO) in glycerol (Slow Fade Light Antifade Kit; Molecular Probes). Cells were imaged (0.35µm slice thickness) using a Zeiss LSM 5 Pascal laser confocal microscope with a x63 objective. To compare different mutants, the same gain, pin-hole and exposure time were used for all channels.

Analysis

All values are reported as mean ±S.E.M. Carbachol- (‘c’ in Fig. 1B inset) and ethanol-induced (‘e’) currents were expressed as a ‘fold’ increase over basal current (‘b’), fold increase = c/b or e/b, respectively. One-way ANOVA followed by a post hoc Dunnett's test was used to test for statistical significance (P < 0.05), using GIRK2 as control. A Bonferroni post hoc test was used to evaluate differences among mutants. In experiments examining the effect of expressing G_ß subunits, we used Student's two-tailed t test on the absolute current levels to compare basal versus coexpression with G_ß subunits. Distance between amino acids in the GIRK1 structure (PDB:1 N9P, Biological Unit) were measured using a Swiss-PDB viewer and displayed using Accelrys Viewerlite 5.0. The G_ß domains were defined based on the following studies. Huang et al. (1995, 1997) narrowed the G_ß binding domains to Q34–I86 in the N-terminal domain and V273–P462 in the C-terminal domain of GIRK1. Kunkel & Peralta (1995) identified T290–Y356 in GIRK1. Ivanina et al. (2003) demonstrated G_ß binding to F181–G254 and, to a lesser extent, G254–P370 of GIRK1 C-terminal domain. In GIRK2, G_ß binds to I46–L96 of the N-terminal and L310–E380 of the C-terminal domain of GIRK2 (Ivanina et al. 2003). He et al. (2002) demonstrated G_ß binding to N253–Y348 of GIRK4. Krapivinsky et al. (1997) identified two peptide sequences, M364–R383 of GIRK1 and S209–R225 of GIRK4, that exhibited potent inhibition of G_ß binding to native GIRK channels.

	Results

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
C-terminal segment of GIRK2 channels implicated in G_ß activation

To localize the G_ß activation site(s) in GIRK2, 21 different chimeras of IRK1 and GIRK2 (using chimera I1G2_97-190 as the backbone) were constructed by systematically replacing 15–40 amino acid segments of IRK1 with the homologous amino acids from GIRK2 (Fig. 1A). I1G2_97-190 contained the hydrophobic core domain (m1-P-m2) from GIRK2 and N- and C-terminal domains from IRK1. I1G2_97-190 was shown previously to be K⁺ selective and not gated by G proteins like IRK1 (Slesinger, 2001). In addition, chimera I1G2_97-190 preserved part of the G protein gate that was localized to the m2 transmembrane domain (Sadja et al. 2001; Yi et al. 2001). Each chimera cRNA was injected into Xenopus oocytes with either the cRNA for the M2 muscarinic receptor (M2R) or G_ß1 and G₂ cRNAs. To evaluate channel function, we examined three parameters of channel activation. First, we measured the activation of current following stimulation of the M2 muscarinic receptor with carbachol (Fig. 1B, ‘c’ in inset); this activation relies on the endogenous G proteins and is G_ß sensitive. To account for possible changes in expression levels in oocytes, we expressed the carbachol-activated current as a function of the agonist-independent (basal) current. Second, we compared the amplitude of basal current (Fig. 1B, ‘b’ in inset) in oocytes coexpressing the chimera with G_ß subunits (+G_ß) with those coexpressing the chimera and M2R receptor. Coexpression of G_ß subunits in oocytes bypasses the endogenous G proteins, leading to the persistent G_ß activation of GIRK channels (Reuveny et al. 1994). We chose to express this as absolute current and used statistical analysis to assess whether G_ß subunits significantly enhanced the basal current. Third, we measured the amplitude of the inward current induced with 200mM ethanol (Fig. 1B, ‘e’ in inset). Ethanol (EtOH) activates GIRK channels but inhibits IRK1 channels (Kobayashi et al. 1999; Lewohl et al. 1999; Zhou et al. 2001). Activation by ethanol does not require functional G proteins and therefore provides an important assessment of channel function for putative G protein-impaired mutants.

The GIRK2/IRK1 chimeras could be classified into three main groups: no current, G_ß sensitive, and G_ß impaired. Generally, chimeras containing the N-terminal domain of IRK1 and part or all of the C-terminal domain of GIRK2 failed to generate currents when expressed in oocytes (data not shown); these and other non-functional chimeras were not studied further (see Methods). Two chimeras (I1G2_1-352 and I1G2_1-390) were G_ß responsive and activated by ethanol, similar to GIRK2 (Fig. 1). The remaining chimeras (e.g. I1G2_1-190–I1G2_1-335) displayed moderate to large basal currents but little or no activation following stimulation of the muscarinic receptor (Fig. 1E). In addition, the basal current was not enhanced by coexpression with G_ß subunits (Fig. 1F). Thus, these chimeras appear to have impaired G_ß sensitivity. The change in G_ß sensitivity occurred between chimeras I1G2_1-335 and I1G2_1-352. To explore whether the agonist-independent current was G_ß sensitive for these two chimeras, we coinjected the cRNA for the C-terminal domain of ßARK1 (ßARK1-ct), which sequesters free G_ß subunits in oocytes (He et al. 1999). A G_ß-sensitive basal current would be expected to be smaller in the presence of ßARK1-ct. ßARK-ct reduced the carbachol-activated current for GIRK2 and I1G2_1-352, indicating suppression of G_ß activity. The basal currents for I1G2_1-335 changed little in the presence of ßARK1-ct, from -6.6 ± 0.6µA (n= 5) to -5.8 ± 0.3µA (n= 5) with ßARK1-ct. Similarly, the basal current for I1G2_1-352 was not affected by ßARK1-ct (-0.45 ± 0.19µA (n= 8) versus-0.46 ± 0.22µA (n= 5) with ßARK1-ct. These results suggest the agonist-independent current is relatively insensitive to G_ß subunits, although we cannot rule out a small component of G protein-dependent activation.

To define the amino acids involved in G_ß activation better, we constructed point mutations in the region that differed between I1G2_1-335 and I1G2_1-352. We focused on amino acids that are conserved among GIRK channels but differ from IRK1 (Fig. 2A). Five amino acids met this criterion, and each was changed from the amino acid in GIRK2 to the corresponding amino acid in IRK1. GIRK2_F388Y, GIRK2_T343F and GIRK2_F348Y all exhibited carbachol-induced currents that were comparable or larger than those of GIRK2 (Fig. 2B and C). Two mutations in GIRK2, GIRK2_L344E and GIRK2_G347H, however, showed dramatically smaller carbachol-induced currents, 0.13-fold and 0.54-fold increases over basal, respectively (compared to a 7.7-fold increase over basal for GIRK2). Despite the small carbachol response, both mutants showed a stimulated basal current when coexpressed with G_ß subunits as well as normal ethanol-induced currents (Fig. 2B–E). If L344E and G347H decrease the sensitivity of the mutant channel to G_ß subunits, then the agonist-activated response may be impaired because there is an insufficient amount of G_ß subunits generated during carbachol stimulation. In contrast, the high levels of G_ß subunits that are present when G_ß subunits are coexpressed may be sufficient to activate the mutant channels.

View larger version (29K): [in this window] [in a new window]   Figure 2.  Mutation of two conserved amino acids in the C-terminal domain of GIRK2 reduces Gß activation A, sequence alignment of the amino acids in GIRK1, GIRK2, GIRK3, GIRK4 and IRK1. Amino acids that are conserved in GIRK family but differ from IRK1 are indicated in bold. Asterisk indicates 100% identity and small filled circles indicate conserved substitutions. B–E, oocytes were injected with cRNA for the indicated channel and with either the cRNA for M2R or Gß subunits (B). Continuous recordings show the response to 0.3µM carbachol (carb), 200mM ethanol (EtOH) and 95mM NaCl for GIRK2L344E, GIRK2G347H and GIRK2EH. Bar graphs show the average carbachol-induced current normalized to the basal current (C), the average ethanol-induced current normalized to the basal current (D) and the average Gß-induced and basal currents (E). Except for F338Y, the carbachol-induced currents for all other mutants are statistically different from GIRK2. None of the EtOH-activated currents is statistically different from GIRK2. All of the +Gß currents are statistically larger than basal (P < 0.05). The increase for GIRK2EH (4-fold), however, is smaller than that for GIRK2 (25-fold). n= 5–49.

ß

ß

ß

ß

Functional studies with G_ß-impaired mutants expressed in HEK-293T cells

In Xenopus oocytes, GIRK channels can coassemble with an endogenous GIRK subunit (XIR) to produce heteromeric channels (Hedin et al. 1996). To eliminate any possible influence of XIR on the G protein sensitivity of the mutant channels studied in oocytes, we examined the G_ß-impaired mutants expressed in HEK-293T cells using whole-cell patch clamp technique. Similar to oocytes, chimera I1G2_1-335 exhibited a large, agonist-independent current that was not increased further with carbachol (Figs 3B and 4A–C). By contrast, I1G2_1-352 showed both carbachol-induced and ethanol-induced currents, like GIRK2 (Fig. 4B and C). Thus, the G_ß sensitivity of chimeras I1G2_1-335 and I1G2_1-352 expressed in HEK-293T paralleled those observed in oocytes. Interestingly, we also observed a change in ethanol activation between chimeras I1G2_1-335 and I1G2_1-352 (Fig. 4C) in HEK-293T cells, which is similar to that reported by Lewohl et al. (1999).

View larger version (33K): [in this window] [in a new window]   Figure 3.  GIRK2EH expressed in HEK-293T cells has reduced carbachol activation but normal ethanol activation HEK-293T cells were transiently transfected with cDNA for the M2R and either GIRK2 (A), I1G21-335 (B), GIRK2EH (C), or GIRK1 plus GIRK2EH (D) cDNA. Whole-cell patch clamp currents were elicited by voltage steps from -120 to 30 mV. Bath solutions contained 20mM KCl (20 K) solution, 20 K + 0.3µM carb, 20 K + 200mM EtOH or 160mM NaCl.

View larger version (32K): [in this window] [in a new window]   Figure 4.  Summary of G protein sensitivity for mutants channels expressed in HEK-293T cells Bar graphs show the agonist-independent (A), the carbachol-induced (B), and the ethanol-induced (C) current density for each mutant. Using the ethanol-activated current as a measure of channel expression, the carbachol-activated current was normalized to the amplitude of ethanol-activated current (D). *Significant difference from GIRK2 (P < 0.05). n= 5–18.

ß

ß

The small basal and ethanol-activated currents for GIRK2_G347H and GIRK2_EH suggested that these mutants might express less efficiently than the other mutants in HEK-293T cells. To examine the surface expression of GIRK2 mutants, it was necessary to engineer an extracellular haemagglutinin (HA) epitope. The presence of the HA tag does not appear to alter the function of GIRK2 but may have some effect on trafficking (Chen et al. 2002; Ma et al. 2002). HEK-293T cells transfected with HA-GIRK2_L344E showed intense membrane staining, which was the same or slightly more intense than that for wild-type HA-GIRK2 (Fig. 5B and C). By contrast, both HA-GIRK2_G347H and HA-GIRK2_EH expressed at lower levels on the membrane surface, though they were clearly detectable above the background staining of untransfected cells (Fig. 5A, D and E). In the presence of the L344E mutation, the G347H mutation appears to reduce the surface expression of the double mutant HA-GIRK2_EH. GIRK2_EH expression, however, was recovered by coexpression with GIRK1 (Fig. 5F), consistent with the whole-cell patch clamp recordings (Fig. 4). If we normalize the carbachol-activated current to the amplitude of ethanol-activated current, which can serve as a measure of functional channels on the membrane surface, then I1G2_1-335, GIRK2_L344E and GIRK2_EH clearly show reduced carbachol activation (Fig. 4D). Taken together, these results suggest that some of the reduction in carbachol-activated current in HEK-293T cells may be due to the lower expression levels for GIRK2_G347H but not for GIRK2_L344E and GIRK2_EH.

View larger version (101K): [in this window] [in a new window]   Figure 5.  Immunostaining of HA-tagged GIRK2 channels expressed in HEK-293T cells HEK-293T cells were transfected with the channel cDNA indicated in the figure. A–F, surface localization was revealed by immunostaining non-permeabilized cells using an Alexa488 conjugated anti-HA antibody. Images were obtained with a laser confocal microscope, using the same gain, slice thickness (0.35µm), pin-hole and exposure time.

G_ß binding to C-terminal domains of GIRK2/IRK1 chimeras

We next examined whether L344 or G347 is important for the biochemical binding of G_ß to the channel. We used a coaffinity precipitation assay to measure the binding of G_ß subunits to fusion proteins containing the C-terminal domain of the channel (Fig. 6A). In contrast to previous studies examining G_ß binding with increasingly smaller fragments of the C-terminal domain of GIRK channels (Huang et al. 1995; He et al. 1999; Ivanina et al. 2003), we constructed GST fusion proteins that contained the entire cytoplasmic C-terminal domain of the chimera or the GIRK2 point mutant (Fig. 6A). As shown previously, the C-terminal domain of GIRK2 but not IRK1 binds G_ß subunits (Huang et al. 1997). Surprisingly, fusion proteins containing the C-terminal domain from the different chimeras all exhibited G_ß binding similar to GIRK2 (Fig. 6B). Chimeras containing either the proximal (GST-I1G2_181-230) or distal (GST-I1G2_353-414) region of GIRK2 exhibited similar G_ß binding that was clearly greater than the G_ß binding to the GST alone or the C-terminal domain of IRK1. By contrast, the G_ß binding to the C-terminal domain of GIRK2_EH appeared slightly reduced (Fig. 6C). To quantify these possible differences in G_ß binding, we measured the optical density (OD) of the G_ß band, divided by the OD for the GST band and then normalized to the G_ß binding for GIRK2 (Fig. 6D). Compared to the full-length C-terminal domain of GIRK2 (GST-G2_191-414), only GST-G2_L344E and GST-I1_173-428 showed significantly less G_ß binding (Fig. 6D). However, G_ß binding to GST-G2_L344E was not statistically different from GST-G2_G347H or GST-G2_EH. Taken together, the electrophysiological and biochemical experiments suggest that L344 plays a major role in G_ß activation and, to a lesser extent, in G_ß binding.

View larger version (38K): [in this window] [in a new window]   Figure 6.  Mutations in ßL–ßM loop of GIRK2 show variable changes in Gß binding In vitro coaffinity precipitation (AP) assays were used to measure the Gß binding to GST fusion proteins containing the C-terminal domain of the wild-type or mutant channel. A, schematic diagram of GST fusion proteins. B, GST fusion proteins (200nM) were mixed with purified bovine brain Gß subunits (40nM). Immuno-blot (IB) using antibody against Gß (upper panel) and, after stripping the blot, against GST (lower panel). All chimeras showed some Gß binding. C, Gß binding assay for GST, GST-I1173-428, GST-G2191-414, GST-G2EH, GST-G2336-414 and GST-G2353-414. D, quantification of Gß binding. The optical density (OD) of the Gß band was divided by the OD of the GST band, and then normalized to the GIRK2 for each blot (n indicated in parentheses). *Statistical difference from GST-G2191-414 (P < 0.05, ANOVA followed by post hoc Bonferroni test). GST-G2L344E is not statistically different from GST-G2G347H or GST-G2EH.

	Discussion

Top Abstract Introduction Methods Results Discussion Supplementary material References

Important role for ßL–ßM loop in G_ß activation

We first evaluated in Xenopus oocytes the ability of mutant GIRK channels to be activated through stimulation of the m2 muscarinic receptor as well as by coexpression with G_ß subunits. In the latter case, we assume that the concentration of free G_ß is significantly higher as compared to the G_ß liberated during stimulation of the m2 muscarinic receptor. This supposition seems justified since the carbachol-activated currents were on average significantly smaller than the basal currents recorded in oocytes coexpressing G_ß subunits. We found that GIRK2 channels containing either the L344E or G347H mutation exhibited dramatically smaller carbachol-activated currents but were still activated by coexpressed G_ß subunits. Combining the two mutations (GIRK2_EH) produced channels that were unresponsive to muscarinic receptor stimulation and now showed little enhancement with coexpressed G_ß subunits. Electrophysiological recordings obtained in HEK-293T cells also supported the conclusion that L344E and, to a lesser extent, G347H, were important for G_ß activation. Interestingly, L344 and G347 are both conserved among the different types of GIRK channels but differ in G protein-insensitive inward rectifiers (see Supplementary Material available online), suggesting an important functional role for these amino acids. Assuming the three-dimensional structure of the GIRK2 cytoplasmic domains is the same as that of GIRK1 (Nishida & MacKinnon, 2002), L344 and G347 would be located in an exposed loop (ßL–ßM) facing the intracellular milieu (see Fig. 7B).

View larger version (40K): [in this window] [in a new window]   Figure 7.  Multiple sites for Gß interaction with GIRK channels: a role for the ßL–ßM loop in Gß activation A, alignment of the Gß binding domains in the N- and C-terminal domains of GIRK channel identified in the following studies: aHuang et al. 1995, 1997; bKunkel & Peralta, 1995; cHe et al. 1999, 2002; dKrapivinsky et al. 1998; eIvanina et al. 2003; and fthis study (see Methods for details). Filled bars (red, green, black): regions binding Gß subunits. Shaded red bar: regions showing less Gß binding. Stippled bars: peptide competing for Gß binding. Arrow marks position of the leucine in the ßL–ßM loop. The GIRK1 sequence included in 3-D structure is indicated by the black line. Three Gß binding domains are drawn to indicate regions of most overlap for Gß binding, along with a region unique to GIRK1 (see text for details). B, three putative Gß segments are highlighted in the GIRK1 structure (Nishida & MacKinnon, 2002). Model shows four subunits arranged around a pore (circle), with two subunits highlighting Gß domains: region 1 (R43–E63, turquoise), region 2 (R190–R219, yellow) and region 3 (E300–P370, red). The amino acid sequence between regions 2 and 3 (green) has also been implicated in Gß binding. Region 4 is unique to GIRK1 and is not present in the structure. Note proximity of the N-terminal domain to the ßL–ßM loop. Two amino acids, L262 (yellow, He et al. 2002) and L333E (red) are highlighted in the structure. C, side view of the same structure. Dashed line indicates approximate position of the cytoplasmic side of the plasma membrane.

ß

ß

ß

ß

The L344 in the ßL–ßM loop of GIRK2 has been implicated previously in other GIRK channels. In a study of GIRK4, He et al. (1999) discovered that L339E in GIRK4 (homologous to GIRK2_L344E) impaired agonist-induced activation, similar to GIRK2_L344E and GIRK2_EH. In addition to a loss of agonist-activated current, GIRK4*_L339E exhibited a large, agonist-independent current that was not increased by coexpression with G_ß subunits (He et al. 1999). The basal current was suppressed by coexpression of ßARK1-ct or G G protein, however, leading He et al. (1999) to postulate that the agonist-induced current and G_ß-dependent basal current are generated by two different G_ß binding sites on the channel; a high affinity site that produces the agonist-independent current and is saturated under basal conditions, and a low affinity G_ß binding site that is occupied following agonist activation. For generating large, agonist-independent currents for homomeric GIRK4 channels, He et al. (1999) introduced a mutation in the pore of GIRK4 (GIRK4*_S143T) to promote the expression of the homomultimer (Chan et al. 1996; He et al. 1999). Although the S143T mutation has no effect on single-channel kinetics, it is unclear how the mutation leads to larger currents. Interestingly, Peleg et al. (2002) found that GIRK channels expressed at high levels in oocytes leads to large, agonist-independent currents with small receptor-activated currents, due to a limiting supply of G subunits. In our study, the expression of GIRK2 in oocytes or HEK-293T cells did not generate an agonist-independent current that was large enough to reliably test the effect of ßARK1-ct. In a recent study by Ivanina et al. (2003), GIRK1_L333E/GIRK2_L344E heteromultimers expressed in oocytes displayed small, agonist-independent and agonist-activated currents, similar to our results. Collectively, these studies provide convincing evidence that the leucine in the ßL–ßM loop plays an important functional role in G_ß activation.

How does mutating the leucine in the ßL–ßM loop account for the change in G_ß activation? One possibility is that the binding of G_ß subunits to the channel is altered. The effect of the leucine-to-glutamate mutation on the biochemical binding of G_ß, however, is more equivocal. He et al. (1999) reported an 60% decrease in G_ß binding to the C-terminal domain of GIRK4_L339E. However, G_ß binding was also reduced for GIRK4_L268I, which had a defective G_ß-dependent basal current (He et al. 2002). Using in vitro translated ³⁵S-labelled G_ß to measure binding to GIRK channels, Ivanina et al. (2003) observed a 30–40% decrease in binding to the C-terminal domain of GIRK1_L333E but no detectable change in G_ß binding to the C-terminal domain of GIRK2_L344E. In our experiments with purified G_ß subunits, L344E exhibited 60% less G_ß binding but the double mutant (GIRK2_EH) and the chimeric channel (I1G2_1-335), which both exhibited defective G_ß activation, were not statistically different from control. One complication to the binding studies is that G_ß subunits bind to multiple regions in the C-terminal domain (see below), thereby potentially masking changes in G_ß binding.

Even with a twofold decrease in G_ß binding, it may appear difficult to reconcile this small change with a major loss of agonist-activated and G_ß-stimulated currents. A limitation to the G_ß binding assay, however, is that G_ß binding is measured in vitro with a fusion protein that lacks the transmembrane domains. If we consider GIRK channels to be allosteric proteins, then the affinity for G_ß subunits may change depending on the state of the channel (Changeux & Edelstein, 1998). Thus, it remains possible that G_ß binding is altered more dramatically in the context of the intact mutant channel. On the other hand, G_ß activation is highly cooperative, requiring the binding of multiple G_ß dimers to the channel (Corey & Clapham, 2001). Thus, a subtle mutation in all four subunits may alter the cooperativity of G_ß activation and lead to reduced G_ß activation. Finally, mutations in the ßL–ßM loop may interfere with the coupling of G_ß binding to the channel's activation gate. Future studies will clarify the link between G_ß binding and channel activation.

G_ß binding sites mapped on the GIRK1 structure

In addition to the ßL–ßM loop, several other regions in the C-terminal domain of GIRK channels have been implicated in G_ß binding. We compared the G_ß binding domains implicated in this and previous studies, and searched for regions of greatest overlap. Based on this criterion, we identified three general regions in GIRK channels important for G_ß binding (Fig. 7A, see Methods for details). Region 1 contains part of the N-terminal domain. Defining the regions in the C-terminal domain was more difficult and somewhat subjective, since G_ß sites appear to be distributed over the entire C-terminal domain. We suggest that two general regions, a proximal (region 2) and middle (region 3) C-terminal segment of GIRK1–4, can account for most of the overlap in G_ß binding sites. Region 3 encompasses the ßL–ßM loop and surrounding amino acids. A fourth region (region 4) may exist that is unique to the distal end of GIRK1. Mapping these putative G_ß binding segments onto the three-dimensional structure of GIRK1 reveals that they are clustered on the outer edge of the tetrameric channel, away from the central pore, and well positioned to interact with G_ß subunits (Fig. 7B). Further refinement of the G_ß sites on GIRK channels will require the structural determination of a complex of G_ß subunits and GIRK cytoplasmic domains, as well as incoporation of the G binding site (Huang et al. 1995) and PIP₂ sites (Huang et al. 1998; Sui et al. 1998).

The propinquity of the ßL–ßM loop to the N-terminal domain suggests a testable model for G_ß activation. The ßL–ßM loop is situated within 4 Å (Q44–G336 distance is 3.6 Å) of the N-terminal domain. Thus, G_ß binding may alter the interaction between the ßL–ßM loop of one subunit with the N-terminal domain of the neighbouring subunit. Strengthening the bonds within the loop and the N-terminal domain might interfere with G_ß activation. Using the Swiss-Pdb Viewer program to model mutations in GIRK1, the side-chain of the glutamate (L333E) could form hydrogen bonds with the backbone of E334, E335 and F337, within the ßL–ßM loop. The side-chain of the histidine (G336H) could form a hydrogen bond with the Q44 located in the N-terminal domain. Based on this model, it may be possible to create a mutation that will rescue the G_ß defect of L344E/G347H. Several lines of evidence support an important role for coupling between the N- and C-terminal domains in other types of inward rectifiers during channel activation. First, the N-terminal domain of GIRK binds G_ß subunits and interacts cooperatively with the C-terminal domain (Huang et al. 1997). Second, Schulte et al. (1998) found that cysteines in the N- and C-terminal domains of ROMK1 (Kir1.1) were modified only in the closed state, indicating a conformational change in the cytoplasmic domains. Finally, mutations in the N-terminal domain of K_ATP channel subunit (Kir6.2) disrupt the binding of the N-terminal domain to the C-terminal domain (Tucker & Ashcroft, 1999).

	Supplementary material

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
The online version of this paper can be found at:

DOI: 10.1113/jphysiol.2003.056101

and contains supplementary material consisting of a figure entitled:

Clustal alignment of rodent Kir family of channels.

This can also be accessed at http://www.blackwellpublishing.com/products/journals/suppmat/tjp/tjp131sm.htm

	Footnotes

 
Authors M. Finley and C. Arrabit contributed equally to this work.

	References

Top Abstract Introduction Methods Results Discussion Supplementary material References

 
Blednov YA, Stoffel M, Chang SR & Harris RA (2001). Potassium channels as targets for ethanol: studies of G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2) null mutant mice. J Pharmacol Exp Ther 298, 521–530.[Abstract/Free Full Text]

Chan KW, Sui JL, Vivaudou M & Logothetis DE (1996). Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K⁺ channel subunit. Proc Natl Acad Sci U S A 93, 14193–14198.[Abstract/Free Full Text]

Changeux J-P & Edelstein S (1998). Allosteric receptors after 30 years. Neuron 21, 959–980.[CrossRef][Medline]

Chen L, Kawano T, Bajic S, Kaziro Y, Itoh H, Art JJ, Nakajima Y & Nakajima S (2002). A glutamate residue at the C terminus regulates activity of inward rectifier K⁺ channels: Implication for Andersen's syndrome. Proc Natl Acad Sci U S A 99, 8430–8435.[Abstract/Free Full Text]

Corey S & Clapham DE (2001). The stoichiometry of Gß binding to G-protein-regulated inwardly rectifying K⁺ channels (GIRKs). J Biol Chem 276, 11409–11413.[Abstract/Free Full Text]

Doupnik CA, Davidson N & Lester HA (1995). The inward rectifier potassium channel family. Curr Opin Neurobiol 5, 268–277.[CrossRef][Medline]

Finley M, Arrabit C, Fowler C & Slesinger PA (2003). Identification of a conserved C-terminal region of Kir3 important for Gß activation. In Biophysical Society 47th Annual Meeting. Biophysical Society, San Antonio, Texas.

Hamill OP, Marty A, Neher E, Sakmann B & Sigworth FJ (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391, 85–100.[CrossRef][Medline]

He C, Yan X, Zhang H, Mirshahi T, Jin T, Huang A & Logothetis DE (2002). Identification of critical residues controlling G protein-gated inwardly rectifying K⁺ channel activity through interactions with the ß subunits of G proteins. J Biol Chem 277, 6088–6096.[Abstract/Free Full Text]

He C, Zhang H, Mirshahi T & Logothetis DE (1999). Identification of a potassium channel site that interacts with G protein ß subunits to mediate agonist-induced signaling. J Biol Chem 274, 12517–12524.[Abstract/Free Full Text]

Hedin KE, Lim NF & Clapham DE (1996). Cloning of a Xenopus laevis inwardly rectifying K⁺ channel subunit that permits GIRK1 expression of IKACh currents in oocytes. Neuron 16, 423–429.[CrossRef][Medline]

Hille B (1992). Ionic Channels of Excitable Membranes. Sinauer Assoc, Sunderland, MA, USA.

Ho IH & Murrell-Lagnado RD (1999). Molecular determinants for sodium-dependent activation of G protein-gated K⁺ channels. J Biol Chem 274, 8639–8648.[Abstract/Free Full Text]

Huang C-L, Feng S & Hilgemann DW (1998). Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gß. Nature 391, 803–806.[CrossRef][Medline]

Huang CL, Jan YN & Jan LY (1997). Binding of the G protein ß subunit to multiple regions of G protein-gated inward-rectifying K⁺ channels. FEBS Lett 405, 291–298.[CrossRef][Medline]

Huang CL, Slesinger PA, Casey PJ, Jan YN & Jan LY (1995). Evidence that direct binding of Gß to the GIRK1 G protein-gated inwardly rectifying K⁺ channel is important for channel activation. Neuron 15, 1133–1143.[CrossRef][Medline]

Inanobe A, Morishige K-I, Takahashi N, Ito H, Yamada M, Takumi T, Nishina H, Takahashi K, Kanaho Y, Katada T & Kurachi Y (1995). Gß directly binds to the carboxyl terminus of the G protein-gated muscarinic K⁺ channel, GIRK1. Biochem Biophys Res Commun 212, 1022–1028.[CrossRef][Medline]

Inanobe A, Yoshimoto Y, Horio Y, Morishige K-I, Hibino H, Matsumoto S, Tokunaga Y, Maeda T, Hata Y, Takai Y & Kurachi Y (1999). Characterization of G-protein-gated K⁺ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra. J Neurosci 19, 1006–1017.[Abstract/Free Full Text]

Ivanina T, Rishal I, Varon D, Mullner C, Frohnwieser-Steinecke B, Schreibmayer W, Dessauer CW & Dascal N (2003). Mapping the Gß-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K⁺ channel. J Biol Chem 278, 29174–29183.[Abstract/Free Full Text]

Jelacic TM, Kennedy ME, Wickman K & Clapham DE (2000). Functional and biochemical evidence for G-protein-gated inwardly rectifying K⁺ (GIRK) channels composed of GIRK2 and GIRK3. J Biol Chem 275, 36211–36216.[Abstract/Free Full Text]

Kennedy ME, Nemec J & Clapham DE (1996). Localization and interaction of epitope-tagged GIRK1 and CIR inward rectifier K⁺ channel subunits. Neuropharmacology 35, 831–839.[CrossRef][Medline]

Kobayashi T, Ikeda K, Kojima H, Niki H, Yano R, Yoshiola T & Kumanishi T (1999). Ethanol opens G-protein-activated inwardly rectifying K⁺ channels. Nat Neurosci 2, 1091–1097.[CrossRef][Medline]

Krapivinsky G, Kennedy ME, Nemec J, Medina I, Krapivinsky L & Clapham DE (1998). G_ß binding to GIRK4 subunit is critical for G protein-gated K⁺ channel activation. J Biol Chem 273, 16946–16952.[Abstract/Free Full Text]

Kubo Y, Baldwin TJ, Jan YN & Jan LY (1993a). Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127–133.[CrossRef][Medline]

Kubo Y, Reuveny E, Slesinger PA, Jan YN & Jan LY (1993b). Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364, 802–806.[CrossRef][Medline]

Kunkel MT & Peralta EG (1995). Identification of domains conferring G protein regulation on inward rectifier potassium channels. Cell 83, 443–449.[CrossRef][Medline]

Lesage F, Duprat F, Fink M, Guillemare E, Coppola T, Lazdunski M & Hugnot JP (1994). Cloning provides evidence for a family of inward rectifier and G-protein coupled K⁺ channels in the brain. FEBS Lett 353, 37–42.[CrossRef][Medline]

Lewohl JM, Wilson WR, Mayfield RD, Brozowski SJ, Morrisett RA & Harris RA (1999). G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action. Nat Neurosci 2, 1084–1090.[CrossRef][Medline]

Liao YJ, Jan YN & Jan LY (1996). Heteromultimerization of G-protein-gated inwardly rectifying K⁺ channel proteins GIRK1 and GIRK2 and their altered expression in weaver brain. J Neurosci 16, 7137–7150.[Abstract/Free Full Text]

Logothetis DE, Kurachi Y, Galper J, Neer EJ & Clapham DE (1987). The ß subunits of GTP-binding proteins activate the muscarinic K⁺ channel in heart. Nature 325, 321–326.[CrossRef][Medline]

Ma D, Zerangue N, Raab-Graham K, Fried SR, Jan YN & Jan LY (2002). Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart. Neuron 33, 715–729.[CrossRef][Medline]

Nishida M & MacKinnon R (2002). Structural basis of inward rectification. Cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution. Cell 111, 957–965.[CrossRef][Medline]

Peleg S, Varon D, Ivanina T, Dessauer CW & Dascal N (2002). Gi controls the gating of the G protein-activated K⁺ channel, GIRK. Neuron 33, 87–99.[CrossRef][Medline]

Petit-Jacques J, Sui JL & Logothetis DE (1999). Synergistic activation of G protein-gated inwardly rectifying potassium channels by the ß subunits of G proteins and Na⁺ and Mg²⁺ ions. J General Physiol 114, 673–684.[Abstract/Free Full Text]

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 ß subunits. Nature 370, 143–146.[CrossRef][Medline]

Sadja R, Smadja K, Alagem N & Reuveny E (2001). Coupling Gß-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels. Neuron 29, 669–680.[CrossRef][Medline]

Schulte U, Hahn H, Wiesinger H, Ruppersberg JP & Fakler B (1998). pH-dependent gating of ROMK (Kir1.1) channels involves conformational changes in both N and C termini. J Biol Chem 273, 34575–34579.[Abstract/Free Full Text]

Signorini S, Liao YJ, Duncan SA, Jan LY & Stoffel M (1997). Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K⁺ channel GIRK2. Proc Natl Acad Sci U S A 94, 923–927.[Abstract/Free Full Text]

Slesinger PA (2001). Ion selectivity filter regulates local anesthetic inhibition of G protein-gated inwardly rectifying K⁺ channels. Biophys J 80, 707–718.[Abstract/Free Full Text]

Slesinger PA, Patil N, Liao YJ, Jan YN, Jan LY & Cox DR (1996). Functional effects of the mouse weaver mutation on G protein-gated inwardly rectifying K⁺ channels. Neuron 16, 321–331.[CrossRef][Medline]