Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.     

Distinct histidine residues control the acid-induced activation and inhibition of the cloned K(ATP) channel

The modulation of K(ATP) channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus.     

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.     

Zn2+ and H+ are coactivators of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in sensory neurons, where they are thought to be involved in pain perception associated with tissue acidosis. They are also expressed in brain. A number of brain regions, like the hippocampus, contain large amounts of chelatable vesicular Zn(2+). This paper shows that Zn(2+) potentiates the acid activation of homomeric and heteromeric ASIC2a-containing channels (i.e. ASIC2a, ASIC1a+2a, ASIC2a+3), but not of homomeric ASIC1a and ASIC3. The EC(50) for Zn(2+) potentiation is 120 and 111 microm for the ASIC2a and ASIC1a+2a current, respectively. Zn(2+) shifts the pH dependence of activation of the ASIC1a+2a current from a pH(0.5) of 5.5 to 6.0. Systematic mutagenesis of the 10 extracellular histidines of ASIC2a leads to the identification of two residues (His-162 and His-339) that are essential for the Zn(2+) potentiating effect. Mutation of another histidine residue, His-72, abolishes the pH sensitivity of ASIC2a. This residue, which is located just after the first transmembrane domain, seems to be an essential component of the extracellular pH sensor of ASIC2a.     

Involvement of histidine residues in proton sensing of ROMK1 channel

ROMK channels are inhibited by intracellular acidification. This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2). Unlike all other amino acids, histidine is titratable at pH 6-7 carrying a positive charge below pH 6. To test the hypothesis that certain histidine residues are engaged in CO(2) and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis. There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO(2). Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO(2) and pH sensing. Mutation of either of them reduced CO(2) and pH sensitivities by 20-50% and approximately 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO(2) sensitivity and caused this mutant channel to respond to only extremely acidic pH. Similar mutations of His-280 had no effect. The role of His-270 in CO(2) and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any functional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.     

Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G(alpha)q family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G(alpha)i/o- and G(alpha)q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G(beta)gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G(alpha)i/o-coupled) and TRH-R1 (G(alpha)q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G(alpha)q family subunits, rather than G(beta)gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G(alpha)q family, and reduced by minigene constructs that disrupt G(alpha)q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G(beta)gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G(alpha)q was reduced by, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G(alpha) subunits of the G(alpha)q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.     

Direct activation of cloned K(atp) channels by intracellular acidosis

ATP-sensitive K(+) (K(ATP)) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K(ATP) changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K(ATP) was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K(ATP). The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K(ATP) in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K(ATP). The pH-dependent activation may enable the K(ATP) to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.     

Modulation of the Kir7.1 potassium channel by extracellular and intracellular pH

Inwardly rectifying K(+) (K(ir)) channels in the apical membrane of the retinal pigment epithelium (RPE) contribute to extracellular K(+) homeostasis in the distal retina by mediating K(+) secretion. Multiple lines of evidence suggest that these channels are composed of Kir7.1. Previously, we showed that native K(ir) channels in bovine RPE are modulated by changes in intracellular pH in the physiological range. In the present study, we used the Xenopus laevis oocyte expression system to investigate the pH dependence of cloned human Kir7.1 channels and several point mutants involving histidine residues in the NH(2) and COOH termini. Kir7.1 channels were inhibited by strong extracellular acidification and modulated by intracellular pH in a biphasic manner, with maximal activity at about intracellular pH (pH(i)) 7.0 and inhibition by acidification or alkalinization. Replacement of histidine 26 (H26) in the NH(2) terminus with alanine eliminated the requirement of protons for channel activity and increased sensitivity to proton-induced inhibition, resulting in maximal channel activity at alkaline pH(i) and smaller whole cell currents at resting pH(i) compared with wild-type Kir7.1. When H26 was replaced with arginine, the pH(i) sensitivity profile was similar to that of the H26A mutant but with the pK(a) shifted to a more acidic value, giving rise to whole cell current amplitude at resting pH(i) that was comparable to that of wild-type Kir7.1. These results indicate that Kir7.1 channels are modulated by intracellular protons by diverse mechanisms and suggest that H26 is important for channel activation at physiological pH(i) and that it influences an unidentified proton-induced inhibitory mechanism.     

Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels

Activation of the heteromeric G protein-gated inwardly rectifying K(+) channel (GIRK) GIRK1 and GIRK4 subunits gives rise to I(KACh), which controls excitability in atrial tissue. Although homomeric GIRK4 channels localize to the plasma membrane and display moderate function, GIRK1 channels fail to localize to the cell surface and do not exhibit significant function as homomers. Using oocytes to express GFP-tagged GIRK1 and GIRK4 and chimeras between these two proteins, we have identified two regions, one in the proximal C terminus and another in the distal N terminus that are critical for their subcellular localization. Replacement of both of these regions in GIRK1 with corresponding regions from GIRK4 was required for efficient expression of GIRK1 on the plasma membrane. Replacement of either region by itself was ineffective. The distal N terminus and proximal C terminus have been previously suggested to play important roles in ER-export and subunit co-assembly respectively in this family of channels. Our data indicate for the first time that both of these regions need to work in concert to mediate efficient targeting of these channels to the plasma membrane.     

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.     

A single histidine residue determines the pH sensitivity of the pacemaker channel HCN2

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels control the rhythmic activity of heart and neuronal networks. The activation of these channels is regulated in a complex manner by hormones and neurotransmitters. In addition it was suggested that the channels may be controlled by the pH of the cytosol. Here we demonstrate that HCN2, a member of the HCN channel family, is directly modulated by the intracellular pH in the physiological range. Protons inhibit HCN2 channels by shifting the voltage dependence of channel activation to more negative voltages. By using site-directed mutagenesis, we have identified a single histidine residue (His-321) localized at the boundary between the voltage-sensing S4 helix and the cytoplasmic S4-S5 linker of the channel that is a major determinant of pH sensitivity. Replacement of His-321 by either arginine, glutamine, or glutamate results in channels that are no longer sensitive to shifts in intracellular pH. In contrast, cAMP-mediated modulation is completely intact in mutant channels indicating that His-321 is not involved in the molecular mechanism that controls modulation of HCN channel activity by cyclic nucleotides. Because His-321 is conserved in all four HCN channels known so far, regulation by intracellular pH is likely to constitute a general feature of both cardiac and neuronal pacemaker channels.     

A novel small-molecule selective activator of homomeric GIRK4 channels

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.     

Titration of tertiapin-Q inhibition of ROMK1 channels by extracellular protons

Tertiapin-Q (TPN(Q)), a honey bee toxin derivative, inhibits inward-rectifier K(+) channels by binding to their external vestibule. In the present study we found that TPN(Q) inhibition of the channels is profoundly affected by extracellular pH. This pH dependence mainly reflects titration of histidine residue 12 in TPN(Q) by extracellular protons, since it largely vanishes when the histidine residue is replaced with alanine. Not surprisingly, this alanine derivative of TPN(Q) binds to the channel with much lower affinity. Quantitative thermodynamic cycle analysis shows that deprotonation of the histidine residue reduces the TPN(Q)-ROMK1 binding energy by 1.6 kcal/mol. To eliminate pH sensitivity but retain high affinity, we derivatized TPN(Q) by replacing histidine 12 with lysine. This derivative-denoted tertiapin-KQ (TPN(KQ))-not only is practically insensitive to extracellular pH but also binds to the channel with even higher affinity than TPN(Q) at extracellular pH 7.6.     

Gbeta residues that do not interact with Galpha underlie agonist-independent activity of K+ channels.

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.     

Requirement of multiple protein domains and residues for gating K(ATP) channels by intracellular pH.

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.     

Two SUR1-specific histidine residues mandatory for zinc-induced activation of the rat KATP channel

Zinc at micromolar concentrations hyperpolarizes rat pancreatic beta-cells and brain nerve terminals by activating ATP-sensitive potassium channels (KATP). The molecular determinants of this effect were analyzed using insulinoma cell lines and cells transfected with either wild type or mutated KATP subunits. Zinc activated KATP in cells co-expressing rat Kir6.2 and SUR1 subunits, as in insulinoma cell lines. In contrast, zinc exerted an inhibitory action on SUR2A-containing cells. Therefore, SUR1 expression is required for the activating action of zinc, which also depended on extracellular pH and was blocked by diethyl pyrocarbonate, suggesting histidine involvement. The five SUR1-specific extracellular histidine residues were submitted to site-directed mutagenesis. Of them, two histidines (His-326 and His-332) were found to be critical for the activation of KATP by zinc, as confirmed by the double mutation H326A/H332A. In conclusion, zinc activates KATP by binding itself to extracellular His-326 and His-332 of the SUR1 subunit. Thereby zinc could exert a negative control on cell excitability and secretion process of pancreatic beta-and alpha-cells. In fact, we have recently shown that such a mechanism occurs in hippocampal mossy fibers, a brain region characterized, like the pancreas, by an important accumulation of zinc and a high density of SUR1-containing KATP.     

G-protein mediated gating of inward-rectifier K+ channels.

G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1-4 (also designated Kir3.1-4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol 4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G-protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G-protein modulation of GIRK channels and their physiological importance in mammals.     

Identification of critical residues controlling G protein-gated inwardly rectifying K(+) channel activity through interactions with the beta gamma subunits of G proteins.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are activated through direct interactions of their cytoplasmic N- and C-terminal domains with the beta gamma subunits of G proteins. By using a combination of biochemical and electrophysiological approaches, we identified minimal N- and C-terminal G beta gamma -binding domains responsible for stimulation of GIRK4 channel activity. Within these domains one N-terminal residue, His-64, and one C-terminal residue, Leu-268, proved critical for G beta gamma-mediated GIRK4 activity. Moreover, mutations at these GIRK4 sites reduced significantly binding of the channel domains to G beta gamma . The corresponding residues in GIRK1 also showed a critical involvement in G beta gamma sensitivity. In GIRK4/GIRK1 heteromers the GIRK4 His-64 and Leu-268 residues showed greater contributions to G beta zeta sensitivity than did the corresponding GIRK1 His-57 and Leu-262 residues. These results identify functionally important channel interaction sites with the beta gamma subunits of G proteins, critical for channel activity.     

Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.     

Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.