An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins.

The PKC family consists of several closely related kinases. These enzymes regulate the function of proteins through the phosphorylation of hydroxyl groups on serines and/or threonines. The selective activation of individual PKC isozymes has proven challenging because of a lack of specific activator molecules. Here, we developed an optogenetic blue light-activated PKC isozyme that harnesses a plant-based dimerization system between the photosensitive cryptochrome-2 (CRY2) and the N terminus of the transcription factor calcium and integrin-binding protein 1 (CIB1) (N-terminal region of the CRY2-binding domain of CIB1). We show that tagging CRY2 with the catalytic domain of PKC isozymes can efficiently promote its translocation to the cell surface upon blue light exposure. We demonstrate this system using PKCε and show that this leads to robust activation of a K+ channel (G protein-gated inwardly rectifying K+ channels 1 and 4), previously shown to be activated by PKCε. We anticipate that this approach can be utilized for other PKC isoforms to provide a reliable and direct stimulus for targeted membrane protein phosphorylation by the relevant PKCs.

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.

The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

G-protein-gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel-phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2).

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel-PIP2 interactions. H2S regulation was attenuated when channel-PIP2 interactions were strengthened and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.

A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels.

Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABAB pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (SEP)-196 and Arg-201, reducing Arg-201 interaction with the sodium-binding site Asp-228. Neutralization of either SEP-196 or Arg-201 leads to a channel with reduced activity and increased sensitivity to PKC inhibition. This study clarifies the role of Ser-196 as an allosteric modulator of PKC inhibition and suggests that the SEP-196/Arg-201 interaction is critical for maintaining maximal channel activity. SIGNIFICANCE STATEMENT: The inwardly rectifying potassium 3.2 (Kir3.2) channel is found principally in neurons that regulate diverse brain functions, including pain perception, alcoholism, and substance addiction. Activation or inhibition of this channel leads to changes in neuronal firing and chemical message transmission. The Kir3.2 channel is subject to regulation by intracellular signals including sodium, G-proteins, ethanol, the phospholipid phosphatidylinositol bis-phosphate, and phosphorylation by protein kinases. Here, we take advantage of the recently published structure of Kir3.2 to provide an in-depth molecular view of how phosphorylation of a specific residue previously thought to be the target of PKC promotes channel gating and acts as an allosteric modulator of PKC-mediated inhibition.

Gα13/PDZ-RhoGEF/RhoA signaling is essential for gastrin-releasing peptide receptor-mediated colon cancer cell migration.

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA-Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.

Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells.

The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices. The limited penetration of the evanescent wave illuminates a narrow specimen field, which facilitates the localization of fluorescently tagged proteins at the cell membrane but not inside of the cell. In addition to constraining the depth of the image, TIRFM also significantly enhances the signal-to-noise ratio, which is particularly valuable in the study of live cells. Here, we detail a protocol for micromirror TIRFM analysis of optogenetically activated protein kinase C-ε in HEK293-T cells, as well as data analysis to demonstrate the translocation of this construct to the cell-surface following optogenetic activation. Graphic abstract.

Optogenetic dephosphorylation of phosphatidylinositol 4,5 bisphosphate in Xenopus laevis oocytes.

Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022).1.

Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP2.

Cardiovascular diseases remain the leading cause of death worldwide. Most deaths are sudden and occur secondary to the occlusion of coronary arteries resulting in a rapid decrease in cellular oxygen levels. Acute hypoxia is proarrhythmic, leading to disordered electrical signals, conduction block, and uncoordinated beating of the myocardium. Although acute hypoxia is recognized to perturb the electrophysiology of heart muscle, the mechanistic basis for the effect has remained elusive, hampering the development of targeted therapeutic interventions. Here, we show that acute hypoxia activates the redox-sensitive SUMO pathway in cardiomyocytes, causing rapid inhibition of the inward-rectifying K+ channel, Kir2.1. We find that SUMOylation decreases the activation of Kir2.1 channels by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). These data provide a mechanistic basis for the proarrhythmic effects of acute hypoxia and offer a framework for understanding the central role of PIP2 in mediating the sequelae of hypoxia and SUMOylation in cardiovascular disease.

Unifying Mechanism of Controlling Kir3 Channel Activity by G Proteins and Phosphoinositides.

The question that started with the pioneering work of Otto Loewi in the 1920s, to identify how stimulation of the vagus nerve decreased heart rate, is approaching its 100th year anniversary. In the meantime, we have learned that the neurotransmitter acetylcholine acting through muscarinic M2 receptors activates cardiac potassium (Kir3) channels via the ßγ subunits of G proteins, an important effect that contributes to slowing atrial pacemaker activity. Concurrent stimulation of M1 or M3 receptors hydrolyzes PIP2, a signaling phospholipid essential to maintaining Kir3 channel activity, thus causing desensitization of channel activity and protecting the heart from overinhibition of pacemaker activity. Four mammalian members of the Kir3 subfamily, expressed in heart, brain, endocrine organs, etc., are modulated by a plethora of stimuli to regulate cellular excitability. With the recent great advances in ion channel structural biology, three-dimensional structures of Kir3 channels with PIP2 and the Gßγ subunits are now available. Mechanistic insights have emerged that explain how modulatory control of activity feeds into a core mechanism of channel-PIP2 interactions to regulate the conformation of channel gates. This complex but beautiful system continues to surprise us for almost 100 years with an apparent wisdom in its intricate design.

Single-cell RT-PCR analysis of GIRK channels expressed in rat locus coeruleus and nucleus basalis neurons.

G protein-coupled inward rectifier potassium channels (GIRK, Kir3) play a crucial role in determining neuronal excitability. Currently, four mammalian GIRK members (GIRK1-4) have been genetically identified. We have been investigating physiological properties of GIRKs in cultured noradrenergic neurons from the locus coeruleus (LC) and cholinergic neurons from the nucleus basalis (NB). Yet, precise information is lacking about which types of GIRK channels are present in these neurons. We performed single-cell RT-PCR on these cultured neurons. In 13 noradrenergic LC neurons, GIRK1, GIRK2, GIRK3, and GIRK4 mRNAs existed in 12, 13, nine, and six neurons, respectively. In six cholinergic NB neurons, GIRK1, GIRK2, GIRK3, and GIRK4 mRNAs existed in six, four, one, and three neurons, respectively. Therefore, GIRK1 and GIRK2 mRNAs are most frequently encountered in both LC and NB neurons.

A computational model predicts that Gßγ acts at a cleft between channel subunits to activate GIRK1 channels.

The atrial G protein (heterotrimeric guanine nucleotide-binding protein)-regulated inwardly rectifying K(+) (GIRK1 and GIRK4) heterotetrameric channels underlie the acetylcholine-induced K(+) current responsible for vagal inhibition of heart rate and are activated by the G protein ßγ subunits (Gßγ). We used a multistage protein-protein docking approach with data from published structures of GIRK1 and Gßγ to generate an experimentally testable interaction model of Gßγ docked onto the cytosolic domains of the GIRK1 homotetramer. The model suggested a mechanism by which Gßγ promotes the open state of a specific cytosolic gate in the channel, the G loop gate. The predicted structure showed that the Gß subunit interacts with the channel near the site of action for ethanol and stabilizes an intersubunit cleft formed by two loops (LM and DE) of adjacent channel subunits. Using a heterologous expression system, we disrupted the predicted GIRK1- and Gßγ-interacting residues by mutation of one protein and then rescued the regulatory activity by mutating reciprocal residues in the other protein. Disulfide cross-linking of channels and Gßγ with cysteine mutations at the predicted interacting residues yielded activated channels. The mechanism of Gßγ-induced activation of GIRK4 was distinct from GIRK1 homotetramers. However, GIRK1-GIRK4 heterotetrameric channels activated by Gßγ displayed responses indicating that the GIRK1 subunit dominated the response pattern. This work demonstrated that combining computational with experimental approaches is an effective method for elucidating interactions within protein complexes that otherwise might be challenging to decipher.

Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex.

Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-ßs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-ß3 bound to activated Gα(q) reveals a conserved module found within PLC-ßs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-ß3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-ß3 subsequently accelerates guanosine triphosphate hydrolysis by Gα(q), causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.

Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain.

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).

Interaction of Galphaq and Kir3, G protein-coupled inwardly rectifying potassium channels.

Activation of substance P receptors, which are coupled to Galpha(q), inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation. We have shown previously that this channel inactivation is not caused by reduction of the phosphatidylinositol 4,5-bisphosphate level in membrane. Moreover, Galpha(q) immunoprecipitates with Kir3.2 (J Physiol 564:489-500, 2005), suggesting that Galpha(q) interacts with Kir3.2. Positive immunoprecipitation, however, does not necessarily indicate direct interaction between the two proteins. Here, the glutathione transferase pull-down assay was used to investigate interaction between Galpha(q) and the K(+) channels. We found that Galpha(q) interacted with N termini of Kir3.1, Kir3.2, and Kir3.4. However, Galpha(q) did not interact with the C terminus of any Kir3 or with the C or N terminus of Kir2.1. TRPC6 is regulated by the signal initiated by Galpha(q). Immunoprecipitation, however, showed that Galpha(q) did not interact with TRPC6. Thus, the interaction between Galpha(q) and the Kir3 N terminus is quite specific. This interaction occurred in the presence of GDP or GDP-AlF(-)(4). The Galpha(q) binding could take place somewhere between residues 51 to 90 of Kir3.2; perhaps the segment between 81 to 90 residues is crucial. Gbetagamma, which is known to bind to N terminus of Kir3, did not compete with Galpha(q) for the binding, suggesting that these two binding regions are different. These findings agree with the hypothesis (J Physiol 564:489-500, 2005) that the signal to inactivate the Kir3 channel could be mainly transmitted directly from Galpha(q) to Kir3.

Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.

Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel.

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 (PIP(2)). SP rapidly - with a half-time of approximately 10 s with intracellular GTPgammaS and approximately 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 PIP(2) 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 PIP(2). A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP(2) 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 PIP(2) theory of channel inhibition. Co-immunoprecipitation reveals that Galpha(q) binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that G(q) activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Galpha(q) to the channel.

Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3.

G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade. The Gbeta/Galpha interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between Gbeta1 and yeast beta (STE4) suggested that the outer strands of blades 1 and 2 of Gbeta1 could be an interaction area between Gbeta1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between beta-strands, substantially reduced GIRK channel activation, suggesting that these residues are Gbeta/GIRK interaction sites. These mutations did not affect the expression of Gbeta1 or its ability to stimulate PLCbeta2. These residues are surface-accessible and located outside Gbeta/Galpha interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of Gbetagamma with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of Gbeta1 impaired the functional interaction of Gbeta1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of Gbeta1 with Galpha as well as all effectors so far tested.

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome.

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.