Homomeric Channels Research Articles

Ion permeation pathway within the internal pore of P2X receptor channels.

P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.

Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

Inhibition of Acid-Sensing Ion Channels by KB-R7943, a Reverse Na+/Ca2+ Exchanger Inhibitor

KB-R7943, an isothiourea derivative, is widely used as a pharmacological inhibitor of reverse sodium-calcium exchanger (NCX). It has been shown to have neuroprotective and analgesic effects in animal models; however, the detailed molecular mechanisms remain elusive. In the current study, we investigated whether KB-R7943 modulates acid-sensing ion channels (ASICs), a group of proton-gated cation channels implicated in the pathophysiology of various neurological disorders, using the whole-cell patch clamp techniques. Our data show that KB-R7943 irreversibly inhibits homomeric ASIC1a channels heterologously expressed in Chinese Hamster Ovary (CHO) cells in a use- and concentration-dependent manner. It also reversibly inhibits homomeric ASIC2a and ASIC3 channels in CHO cells. Both the transient and sustained current components of ASIC3 are inhibited. Furthermore, KB-R7943 inhibits ASICs in primary cultured peripheral and central neurons. It inhibits the ASIC-like currents in mouse dorsal root ganglion (DRG) neurons and the ASIC1a-like currents in mouse cortical neurons. The inhibition of the ASIC1a-like current is use-dependent and unrelated to its effect on NCX since neither of the other two well-characterized NCX inhibitors, including SEA0400 and SN-6, shows an effect on ASIC. Our data also suggest that the isothiourea group, which is lacking in other structurally related analogs that do not affect ASIC1a-like current, may serve as a critical functional group. In summary, we characterize KB-R7943 as a new ASIC inhibitor. It provides a novel pharmacological tool for the investigation of the functions of ASICs and could serve as a lead compound for developing small-molecule drugs for treating ASIC-related disorders.

Diverse functions of the inward-rectifying potassium channel Kir5.1 and its relationship with human diseases.

The inward-rectifying potassium channel subunit Kir5.1, encoded by Kcnj16, can form functional heteromeric channels (Kir4.1/5.1 and Kir4.2/5.1) with Kir4.1 (encoded by Kcnj10) or Kir4.2 (encoded by Kcnj15). It is expressed in the kidneys, pancreas, thyroid, brain, and other organs. Although Kir5.1 cannot form functional homomeric channels in most cases, an increasing number of studies in recent years have found that the functions of this subunit should not be underestimated. Kir5.1 can confer intracellular pH sensitivity to Kir4.1/5.1 channels, which can act as extracellular potassium sensors in the renal distal convoluted tubule segment. This segment plays an important role in maintaining potassium and acid-base balances. This review summarizes the various pathophysiological processes involved in Kir5.1 and the expression changes of Kir5.1 as a differentially expressed gene in various cancers, as well as describing several other disease phenotypes caused by Kir5.1 dysfunction.

A LRRC8 chimera with native functional properties is a heptamer with a large lipid-blocked pore.

The volume-regulated anion channel VRAC mediates volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with an undefined, variable, and experimentally uncontrollable stoichiometry. The heteromeric structure of VRACs greatly complicates structure-function analyses. We circumvented this serious limitation by developing homomeric LRRC8 chimeric channels that recapitulate the functional properties of native heteromeric VRACs. Cryo-EM structures of abnormally functioning LRRC8A and non-functional LRRC8D homomeric channels demonstrated that they have a narrow-pore hexameric structure. In striking contrast, the 8C-8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A is a heptamer of asymmetrically arranged subunits. Five conformationally distinct structures from a single dataset exhibit diverse subunit arrangements and interactions, especially in the cytoplasmic LRR domains. The narrowest region of the 8C-8A(IL125) pore has a solvent-accessible radius of 4.7 Å. This limiting radius is similar to that estimated for native VRACs and is more than twice that of the limiting radii of homohexameric LRRC8A and LRRC8D channels. The 8C-8A(IL125) chimera is strongly inhibited by DCPIB, similar to that of native VRACs. In contrast, homohexameric LRRC8A channels exhibit grossly abnormal DCPIB pharmacology indicating a non-native pore structure. Within the 8C-8A(IL125) pore, we observe two layers of lipids running parallel to the membrane plane. Gaps between protomers are occupied by lipid molecules providing a potential route for lipids to move in and out of the channel pore. Like native VRACs, 8C-8A(IL125) chimeric channels are activated strongly by cell swelling and low intracellular ionic strength. We suggest that VRACs are heptamers similar to closely related pannexin channels and that movement of lipids into and out of the channel pore plays a central role in channel gating.

Heteromeric LRRC8 channel assemblies and their impact on channel activity.

Volume regulated anion channels (VRAC) are activated in response to the osmotic swelling of vertebrate cells to catalyze the efflux of anions and small osmolytes. VRACs are multimeric channels that are composed of leucine-rich repeat-containing 8 (LRRC8) proteins, a protein family encompassing five paralogues in mammals (LRRC8A-E). Endogenous VRACs are known to form heteromeric assemblies of LRRC8A with at least one further LRRC8 isoform. Until recently, only structures of homomeric LRRC8 channels were described, while the structures of heteromeric channels remained elusive. Combining results from targeted quantification mass spectrometry and cryoEM with proteins obtained from heterologous overexpression, we investigated the subunit stoichiometry and structure of heteromeric LRRC8A/C channels. For these proteins, we observed a ratio of LRRC8A:C subunits of 2:1, which was confirmed in cryo-EM structures where LRRC8A and LRRC8C subunits cluster as pairs in a hexameric protein assembly. This subunit arrangement increases the pore size in LRRC8A/C channels compared to homomeric LRRC8A. Moreover, we found a tight interaction between LRR domains of LRRC8A subunit pairs of heteromers, reflecting a similar arrangement observed in homomeric LRRC8A. Conversely, the same parts of LRRC8C subunits were not resolved, indicating increased flexibility of their cytosolic regions. The structure thus suggests that the LRRC8C subunits perturb the tight packing of the LRRC8A subunits in heteromeric channels. Similar destabilizing effects were previously observed in LRRC8A homomers binding synthetic nanobodies with activating phenotype. There the binders induced a destabilization of the LRR domain leading to an increase of the open probability of homomeric channels. Collectively, our results suggest that the incorporation of LRRC8C in heteromeric LRRC8A/C channels destabilizes the tight packing of LRRC8A subunits to improve the activation properties of the channels.

Modulation of heteromeric Kv1 channels by transmembrane lectins and redox.

Voltage-gated potassium channels regulate cellular electrical activity and are influenced by a variety of stimuli including redox and accessory proteins. Kv1.2 channels are exquisitely sensitive to reducing agents, which promote an ‘inhibited’ gating mode characterized by a large depolarizing shift of voltage-dependence, decelerated activation kinetics, and use-dependent activation. Similarly, overexpression of Kv1.2 channels with transmembrane lectin LMAN2 recapitulates these functional outcomes. Kv1 family subunits assemble as heterotetramers with potential tremendous diversity of function and sensitivity. It is unclear whether or how redox or LMAN2 sensitivity of Kv1.2 influences other insensitive Kv1 subtypes when assembled in heteromeric channels. In this study, we tested the redox and LMAN2 sensitivity of a series of homomeric Kv1 and tandem-linked Kv1.2-1.X heteromeric channels. We report that among homomeric channels, redox and LMAN2 sensitivity is exclusive to Kv1.2, while in Kv1.2-containing heteromeric channels, this sensitivity persists. Redox and LMAN2 decelerated the activation kinetics of Kv1.2-containing channels but had no effect on the deactivation kinetics. Interestingly, some Kv1 subtypes (notably Kv1.1 and Kv1.4) appear to be more sensitive than others to Kv1.2-mediated modulation. In summary, these data indicate that Kv1.2 acts as an adaptor subunit capable of recruiting sensitivity to redox and certain accessory proteins. These findings broaden our understanding of the mechanism of modulation of heteromeric voltage-gated potassium channels.

Montelukast activates BK channels associated with the β4 accessory subunit and emerges as a pharmacological tool to reduce hippocampal excitability.

Different β accessory subunits are associated with the large conductance voltage- and Ca2+- dependent K+ channel (BK) in a tissue-specific manner. In particular, BK channel/β4 subunits complexes (α/β4) control neuronal excitability in granule cells of the hippocampal dentate gyrus (GCDG) and, β4 deficient mice display a temporal lobe epilepsy (TLE). Thus, compounds that increase α/β4 activity are of interest for improving epilepsy treatment. Then, based on the β-dependent activating effect of leukotrienes on BK channel, we explore the effect of Montelukast (MTK) on this channel heterologously expressed in HEK cells with or without its β accessory subunits (β1, β2, and β4). MTK is a CysLT1 receptor antagonist used as an anti-asthmatic drug that could share the affinity properties with the leukotrienes. Using the patch-clamp technique we found that MTK shifts the activation curves of α/β1 and α/β4 to more hyperpolarized voltages in a concentration-dependent manner (300 nM MTK: ΔV1/2 (α/β1)= −54.4 ± 8.4 mV; n=6; p<0.05 and ΔV1/2 (α/β4)= −36 ± 13.5 mV; n=5; p<0.05) without affecting α/β2 channel and, only at concentrations greater than 1 µM, slightly shifts the homomeric BK channel curve (ΔV1/2= −10.8 ± 3.7 mV; n=3; p<0.05). MTK delays α/β4 deactivation, suggesting an open channel stabilization (1 µM MTK (−60 mV): τMTK= 27.6 ± 3.7 ms vs. τcontrol= 2.0 ± 0.3 ms n= 5, p<0.05). Finally, as a promising result, we observed that 1 µM MTK decreases the number of action potential spikes induced by 140 pA injection in GCDG from mouse brain slices (58 ± 8%, n=3, p<0.05). Our results indicate that MTK directly activates the α/β4 channel and suggest that it could reduce GCDG excitability and may be a putative new treatment for TLE.

Modulation of delayed rectifying K+ currents by silent Kv subunits.

Voltage-gated potassium channels (VGKCs) play fundamental roles in neurobiology; they are a major hub for potential modulation of cell excitability and health. VGKCs are formed by homotetramerization of K+ channels subunits (Kv1-12). Kv2 channels are the major cause of the delayed rectifying K+ currents (IKdr) that tune neuronal firing frequency. Uniquely, Kv2 subunits can co-assemble with members of four other families of Kv (Kv5-6, 8-9). These other Kvs are named silent since they do not form functional channels themselves as homomeric VGKCs. Heteromeric co-assemblies (hetVGKCs) form biophysically distinct channels that modify IKdr and therefore determine neuronal excitability. More than that, the expression of silent subunits suppresses the overall expression of Kv2 subunits, affecting the abundancy of homomeric Kv2 channels and heteromeric channels containing Kv2 subunits. To date, no established mechanism explains how silent Kv subunits interfere with hetVGKCs gating processes or how the mechanism of suppression works. Here, we used Kv6.4 as archetypal silent subunit expressed together with Kv2.1 in HEK cells. Keeping the total amount of Kv2.1 plasmid constant, we found a significant decrease in the magnitude of currents with increasing Kv6.4 dose (ANOVA, p<0.05). To more cleanly assess biophysical differences between homo- and heterotetramers, we generated a Kv2.1-Kv6.4 dimer and compared currents recorded from cells transfected with Kv2.1 plasmid only vs those transfected with dimer plasmid only. We found a significant hyperpolarizing shift in inactivation V50 (−79.6 mV vs −53.5 mV, t-test, p=0.0001), significantly larger activation slope (20.7 vs 14.9, t-test, p=0.0014) and faster activation kinetics (2-way ANOVA, p=0.0354) for the dimer currents compared to Kv2.1-only currents. These results highlight several ways in which silent subunits modify the function of Kv2.1 currents.

KCNQ channels open with at least two S4 activation.

Voltage-gated potassium KCNQ2/3 channels mediates the M-current, one of the major potassium currents throughout the central and peripheral nervous systems. KCNQ channels regulate the resting membrane potential, shape action potentials, and impede repetitive neuronal firing. KCNQ channels have six transmembrane segments (S1-S6) that form functional tetramers. The S5-S6 of the four subunits form the pore that is flanked by the four voltage-sensing domains (S1-S4). In response to membrane depolarization the voltage sensor (S4) moves to facilitate potassium conductance. However, the number of voltage-sensor activation required to open the pore remains unknown. Here, we use voltage-clamp fluorometry (VCF) and cysteine accessibility approaches to test for concerted or individual S4 movement required to open KCNQ2/3 channels expressed in Xenopus oocytes. We use the nonconducting E2R mutation in the S2 segment of KCNQ2/3, assumed to restrict voltage sensor movement into activated conformations via electrostatic repulsion with S4 charges, to demonstrate that a concerted motion of all four S4s is not required to open KCNQ channels. VCF shows that homomeric labeled-E2R channels are non-conducting but exhibit voltage-dependent fluorescence signals. Tandem constructs containing labeled-KCNQ and E2R in different subunits (to restrain two S4s in the resting state), render KCNQ2-like currents and a robust voltage-dependent fluorescence signal. In contrast, dimers containing wt-KCNQ and labeled -E2R in the same subunit yield ionic currents with negligible voltage-dependent fluorescence signal. The data suggest that KCNQ channels do not require a concerted movement of all four S4 subunits, but rather that channel opening can occur with at least two S4 movement.

Electrophysiological properties of TRPC1/4 heteromer determined by its pore residues.

Transient receptor potential canonical 1 (TRPC1) is widely expressed in various mammalian tissues and is involved in many physiological functions. It is known to heteromerize with TRPC4 to form a cationic channel, and it induces significant changes in channel characteristics. The huge differences in channel characteristics in the physiological environment indicates the importance of channel regulation in many tissues expressing both TRPC1 and TRPC4. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4, compared their homology, and investigated how they contribute to the electrophysiological characteristics of the ion channel. We observed changes in G-V curve and I-V curve with mutations of the pore region and demonstrated that TRPC4 selectivity filter mutants lost sensitivity to G protein mediated activation. Moreover, lower gate mutants of TRPC4 exhibited diminished calcium permeability compared to wild type channels. Through chimeric channels where we substituted the pore region of TRPC1 with that of TRPC4, we were able to determine regions that are critical in producing the double-rectifying I-V curve of the homomeric TRPC4 channel.

Oligomeric structure of LRRC8/VRAC channels: Arguments for a heptameric conformation.

LRRC8 proteins form large-pore heteromeric volume regulated anion/organic solute channels, VRACs, comprising the LRRC8A subunit and another LRRC8 paralog. Heterologous expression of LRRC8A alone gives rise to homomeric channels with non-physiological properties. Purified LRRC8A protein forms narrow-pore homohexameric channels. Recent cryo-EM structures of purified LRRC8A+LRRC8C proteins revealed a narrow-pore heterohexameric structure with stoichiometries of 5A:1C and 4A:2C. We utilize a chimeric protein approach to define structure-function relationships of LRRC8/VRAC channels. A LRRC8 chimera comprising LRRC8C with 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A, 8C-8A(IL125), gives rise to homomeric channels with normal functional properties. Purified 8C-8A(IL125) protein forms a homoheptameric channel with a large pore diameter similar to that of native LRRC8/VRACs. DCPIB is a LRRC8/VRAC inhibitor. 8C-8A(IL125) homomeric and LRRC8A+LRRC8C heteromeric channels exhibit native DCPIB pharmacology. In contrast, the DCPIB pharmacology of LRRC8A homohexameric channels is grossly abnormal. A chimeric channel comprising LRRC8A and the first extracellular loop (EL1) of LRRC8C, 8A-8C(EL1), has similar abnormal DCPIB pharmacology. Purified 8A-8C(EL1) protein also forms narrow-pore homohexameric channels. An EL1 arginine residue unique to LRRC8A, R103, was proposed to participate in DCPIB inhibition. The R103F mutation greatly reduces DCPIB inhibition of LRRC8A homohexameric channels but has no effect on LRRC8A(R103F)+LRRC8C heteromers. The equivalent EL1 mutation, L105F, also reduces DCPIB inhibition of 8A-8C(EL1) homohexameric channels. In contrast, the L105F mutation has no effect on DCPIB inhibition of 8C-8A(IL125) homoheptameric channels. LRRC8 channels most likely evolved from pannexins, which have a well-established heptameric structure. Based on our functional and structural data and the evolutionary origin of LRRC8 proteins, we propose that native LRRC8/VRAC channels have a heteroheptameric configuration that gives rise to a large pore diameter that accounts for their high organic solute permeability.

Evolutionary conservation of Zinc-Activated Channel (ZAC) functionality in mammals: a range of mammalian ZACs assemble into cell surface-expressed functional receptors.

In contrast to the other pentameric ligand-gated ion channels in the Cys-loop receptor superfamily, the ZACN gene encoding for the Zinc-Activated Channel (ZAC) is exclusively found in the mammalian genome. Human ZAC assembles into homomeric cation-selective channels gated by Zn2+, Cu2+ and H+, but the function of the receptor in human physiology is presently poorly understood. In this study, the degree of evolutionary conservation of a functional ZAC in mammals was probed by investigating the abilities of a selection of ZACs from 10 other mammalian species than human to be expressed at the protein level and assemble into cell surface-expressed functional receptors in mammalian cells and in Xenopus oocytes. In an enzyme-linked immunosorbent assay, transient transfections of tsA201 cells with cDNAs of hemagglutinin (HA)-epitope-tagged versions of these 10ZACs resulted in robust total expression and cell surface expression levels of all proteins. Moreover, injection of cRNAs for 6 of these ZACs in oocytes resulted in the formation of functional receptors in two-electrode voltage-clamp recordings. The ZACs exhibited robust current amplitudes in response to Zn2+ (10mM) and H+ (pH 4.0), and the concentration-response relationships displayed by Zn2+ at these channels were largely comparable to that at human ZAC. In conclusion, the findings suggest that the functionality of ZAC at the molecular level may be conserved throughout mammalian species, and that the channel thus may govern physiological functions in mammals, including humans.

Subcellular Localization of Homomeric TASK3 Channels and Its Presumed Functional Significances in Trigeminal Motoneurons

Somatic expressions of either heteromeric TASK1/3 or homomeric TASK1/1 channels have been reported in various neurons, while expression of homomeric TASK3/3 channels has been re-ported only in dendrites. However, it is not known why homomeric TASK3/3 channels are hardly seen in somata of CNS neurons. Given the absence of somatic TASK3/3 channels, it should be clarified why dendritic expression of TASK3/3 channels is inevitable and necessary and how differentially distributed TASK1/1 and TASK3/3 channels play roles in soma-to-dendritic integration. Here, we addressed these questions. We found that TASK3-transfected HEK293 cells showed decreases in cell volume after being transferred from the cultured medium to HEPES Ringer, suggesting that expressions of TASK3 channels in cell bodies cause an osmolarity problem. Using TASK1- and TASK3-transfected oocytes, we also found that cGMP application slightly suppressed TASK3 currents while it largely enhanced TASK1 currents, alleviating the difference between TASK1 and TASK3 currents at physiological pH. As larger motoneurons have extensive dendritic trees while smaller motoneurons have poor ones, cGMP could integrate Ia-EPSPs to recruit small and large motoneurons synchronously by differentially modulating TASKI and TASK3 channels which were complementary distributed in soma and dendrites of motoneurons in the dorsolateral part of the trigeminal motor nucleus.

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)] produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

Intoxicating effects of alcohol depend on acid-sensing ion channels.

Persons at risk for developing alcohol use disorder (AUD) differ in their sensitivity to acute alcohol intoxication. Alcohol effects are complex and thought to depend on multiple mechanisms. Here, we explored whether acid-sensing ion channels (ASICs) might play a role. We tested ASIC function in transfected CHO cells and amygdala principal neurons, and found alcohol potentiated currents mediated by ASIC1A homomeric channels, but not ASIC1A/2 A heteromeric channels. Supporting a role for ASIC1A in the intoxicating effects of alcohol in vivo, we observed marked alcohol-induced changes on local field potentials in basolateral amygdala, which differed significantly in Asic1a-/- mice, particularly in the gamma, delta, and theta frequency ranges. Altered electrophysiological responses to alcohol in mice lacking ASIC1A, were accompanied by changes in multiple behavioral measures. Alcohol administration during amygdala-dependent fear conditioning dramatically diminished context and cue-evoked memory on subsequent days after the alcohol had cleared. There was a significant alcohol by genotype interaction. Context- and cue-evoked memory were notably worse in Asic1a-/- mice. We further examined acute stimulating and sedating effects of alcohol on locomotor activity, loss of righting reflex, and in an acute intoxication severity scale. We found loss of ASIC1A increased the stimulating effects of alcohol and reduced the sedating effects compared to wild-type mice, despite similar blood alcohol levels. Together these observations suggest a novel role for ASIC1A in the acute intoxicating effects of alcohol in mice. They further suggest that ASICs might contribute to intoxicating effects of alcohol and AUD in humans.

Recent insights into gap junction biogenesis in the cochlea.

In the cochlea, connexin 26 (Cx26) and connexin 30 (Cx30) co-assemble into two types of homomeric and heteromeric gap junctions between adjacent non-sensory epithelial cells. These channels provide a mechanical coupling between connected cells, and their activity is critical to maintain cochlear homeostasis. Many of the mutations in GJB2 or GJB6, which encode Cx26 and Cx30 in humans, impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. Thus, deciphering the connexin trafficking pathways in situ should represent a major step forward in understanding the pathogenic significance of many of these mutations. A growing body of evidence now suggests that Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanisms. Here, we review the most recent advances that have been made toward unraveling the biogenesis and stability of these gap junctions in the cochlea.

Expression of p11 and heteromeric TASK channels in mouse adrenal cortical cells and H295R cells

TWIK-related acid-sensitive K+ (TASK) channels are thought to contribute to the resting membrane potential in adrenal cortical (AC) cells. However, the molecular identity of TASK channels in AC cells have not yet been elucidated. Thus, immunocytochemical and molecular biological approaches were employed to investigate the expression and intracellular distribution of TASK1 and TASK3 in mouse AC cells and H295R cells derived from human adrenocortical carcinoma. Immunocytochemical study revealed that immunoreactive materials were mainly located in the cytoplasm for TASK1 and at the cell periphery for TASK3 in mouse AC cells. A similar pattern of localization was observed when GFP-TASK1 and GFP-TASK3 were exogenously expressed in H295R cells. In addition, p11 that is known to suppress the endoplasmic reticulum exit of TASK1 was localized in the cytoplasm in mouse AC and H295R cells, but not in adrenal medullary cells. Proximity ligation assay (PLA) suggested formation of heteromeric TASK1–3 channels that were found predominantly in the cytoplasm and weakly at the cell periphery. A similar distribution was observed following exogenous expression of tandem TASK1–3 channels in H295R cells. When stimulated by angiotensin II, however, tandem TASK1–3 channels were present mainly in the cytoplasm in all H295R cells. In contrast to that in H295R cells, tandem channels were exclusively located at the cell periphery in all non-stimulated and exclusively in the cytoplasm in stimulated PC12 cells, respectively. From these results, we conclude that TASK1 proteins are present mainly in the cytoplasm and minimally at the cell periphery as a heteromeric channel with TASK3, whereas the majority of TASK3 is at the cell periphery as homomeric and heteromeric 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.

Novel Anosmia‐Inducing Compounds for Environmentally Friendly Mosquito Vector Control: Structural Determinants of ORco Ligands Antagonizing Odorant Receptor Function

Insect repellents are important means of personal protection against bites from mosquitoes carrying various pathogens. Odorant‐dependent behaviours are triggered by odor ligands bound to odorant receptors. Insect odorant receptors are heterotetrameric ligand‐gated cation channels composed of an obligatory subunit, ORco, and one of many variable subunits, ORx, to which odorant ligands bind. Upon expression ex vivo, ORco forms homomeric channels gated by specific ligands acting as channel agonists. In past studies, we identified ligands of natural origin that bind to the African malaria mosquito vector Anopheles gambiae ORco (AgamORco). Some of these ligands function as specific ORco channel antagonists, orthosteric or allosteric relative to a predicted ORco agonist binding site, and cause severe inhibition of the olfactory function in a variety of mosquito species. Given that ORco is evolutionarily conserved, we hypothesized that it should be feasible to develop an AgamORco‐based screening platform allowing fast identification of new ORco ligands acting as mosquito odorant receptor antagonists causing anosmia‐like deficits to mosquitoes.We are now reporting on the compilation of common structural features of previously identified orthosteric AgamORco antagonists and generation of a ligand‐based pharmacophore. In silico screening of a collection of volatile compounds of natural origin resulted in identification of several AgamORco ligand hits. Parallel cell‐based screening of the same compounds against AgamORco was also employed in order to identify ligands acting as AgamORco antagonists. The screening resulted in identification of several ligands inhibiting AgamORco channel function ex vivo by at least 40% and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. To test the pharmacophore model for binding site specificity, binding competition assays were undertaken, which distinguished the identified antagonists into orthosteric and allosteric ones. Direct comparisons of the pharmacophore predictions with the results of the cell‐based screen revealed that the pharmacophore predicted correctly 100% of the orthosteric antagonists and none of the allosteric ones. These results confirm the pharmacophore's value for in silico prediction of AgamORco orthosteric ligands. Upon combination with the AgamORco‐specific cell‐based screening platform, the pharmacophore provides a valuable tool for fast identification of multiple, ecologically friendly vector control agents. Finally, to confirm the predicted AgamORco agonist binding site and determine the binding orientation of orthosteric antagonists in it, we are assessing the interactions of these ligands with specific amino acids in the AgamORco binding pocket by site‐specific mutagenesis.