Ion Permeation Pathway Research Articles

Transmembrane channel-like 4 and 5 proteins at microvillar tips are potential ion channels and lipid scramblases.

Microvilli-membrane bound actin protrusions on the surface of epithelial cells-are sites of critical processes including absorption, secretion, and adhesion. Increasing evidence suggests microvilli are mechanosensitive, but underlying molecules and mechanisms remain unknown. Here, we localize transmembrane channel-like proteins 4 and 5 (TMC4 and 5) and calcium and integrin binding protein 3 (CIB3) to microvillar tips in intestinal epithelial cells, near glycocalyx insertion sites. We find that TMC5 colocalizes with CIB3 in cultured cells and that a TMC5 fragment forms a complex with CIB3 in vitro. Homology and AlphaFold2 models reveal a putative ion permeation pathway in TMC4 and 5, and molecular dynamics simulations predict both proteins can conduct ions and perform lipid scrambling. These findings raise the possibility that TMC4 and 5 interact with CIB3 at microvillar tips to form a mechanosensitive complex, akin to TMC1 and 2, and CIB2 and 3, within the mechanotransduction channel complex at the tips of inner ear stereocilia.

Discovery of Selective Inhibitors for the Lysosomal Parkinson's Disease Channel TMEM175.

TMEM175 is a lysosomal potassium and proton channel that is associated with the development of Parkinson's disease. Advances in understanding the physiological roles of TMEM175 have been hampered by the absence of selective inhibitors, and studies involving genetic perturbations have yielded conflicting results. Here, we report the discovery and characterization of the first reported TMEM175-selective inhibitors, 2-phenylpyridin-4-ylamine (2-PPA), and AP-6. Cryo-EM structures of human TMEM175 bound by 2-PPA and AP-6 reveal that they act as pore blockers, binding at distinct sites in the pore and occluding the ion permeation pathway. Acute inhibition of TMEM175 by 2-PPA or AP-6 increases the level of lysosomal macromolecule catabolism, thereby accelerating macropinocytosis and other digestive processes. These inhibitors may serve as valuable tools to study the roles of TMEM175 in regulating lysosomal function and provide useful templates for future therapeutic development in Parkinson's disease.

The role of the STAS domain in SLC26A9 for chloride ion transporter function

The anion exchanger solute carrier family 26 (SLC26)A9, consisting of the transmembrane (TM) domain and the cytoplasmic STAS domain, plays an essential role in regulating chloride transport across cell membranes. Recent studies have indicated that C-terminal helices block the entrance of the putative ion transport pathway. However, the precise functions of the STAS domain and C-terminal helix, as well as the underlying molecular mechanisms governing the transport process, remain poorly understood. In this study, we performed molecular dynamics simulations of three distinct models of human SLC26A9, full-length, STAS domain removal (ΔSTAS), and C-terminus removal (ΔC), to investigate their conformational dynamics and ion-binding properties. Stable binding of ions to the binding sites was exclusively observed in the ΔC model in these simulations. Comparing the full-length and ΔC simulations, the ΔC model displayed enhanced motion of the STAS domain. Furthermore, comparing the ΔSTAS and ΔC simulations, the ΔSTAS simulation failed to exhibit stable ion bindings to the sites despite the absence of the C-terminus blocking the ion transmission pathway in both systems. These results suggest that the removal of the C-terminus not only unblocks the access of ions to the permeation pathway but also triggers STAS domain motion, gating the TM domain to promote ions’ entry into their binding site. Further analysis revealed that the asymmetric motion of the STAS domain leads to the expansion of the ion permeation pathway within the TM domain, resulting in the stiffening of the flexible TM12 helix near the ion-binding site. This structural change in the TM12 helix stabilizes chloride ion binding, which is essential for SLC26A9’s alternate-access mechanism. Overall, our study provides new insights into the molecular mechanisms of SLC26A9 transport and may pave the way for the development of novel treatments for diseases associated with dysregulated ion transport.

Structural insights into ion selectivity and transport mechanisms of Oryza sativa HKT2;1 and HKT2;2/1 transporters.

Plant high-affinity K+ transporters (HKTs) play a pivotal role in maintaining the balance of Na+ and K+ ions in plants, thereby influencing plant growth under K+-depleted conditions and enhancing tolerance to salinity stress. Here we report the cryo-electron microscopy structures of Oryza sativa HKT2;1 and HKT2;2/1 at overall resolutions of 2.5 Å and 2.3 Å, respectively. Both transporters adopt a dimeric assembly, with each protomer enclosing an ion permeation pathway. Comparison between the selectivity filters of the two transporters reveals the critical roles of Ser88/Gly88 and Val243/Gly243 in determining ion selectivity. A constriction site along the ion permeation pathway is identified, consisting of Glu114, Asn273, Pro392, Pro393, Arg525, Lys517 and the carboxy-terminal Trp530 from the neighbouring protomer. The linker between domains II and III adopts a stable loop structure oriented towards the constriction site, potentially participating in the gating process. Electrophysiological recordings, yeast complementation assays and molecular dynamics simulations corroborate the functional importance of these structural features. Our findings provide crucial insights into the ion selectivity and transport mechanisms of plant HKTs, offering valuable structural templates for developing new salinity-tolerant cultivars and strategies to increase crop yields.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

Ruthenium red: Blocker or antagonist of TRPV channels?

Ruthenium red (RR) is a widely used inhibitor of Transient Receptor Potential (TRP) cation channels and other types of ion channels. Although RR has been generally accepted to inhibit TRP channels by physically blocking the ion permeation pathway, recent structural evidence suggests that it might also function as an antagonist, inducing conformational changes in the channel upon binding that result in closure of the pore. In a recent manuscript published in EMBO Reports, Ruth A. Pumroy and collaborators solve structures of TRPV2 and TRPV5 channels in the presence and absence of activators and RR. The data sheds light on the mechanism of inhibition by RR, while also opening new questions for further investigation.

Redox-Regulated Synthetic Channels: Enabling Reversible Ion Transport by Modulating the Ion-Permeation Pathway.

Natural redox-regulated channel proteins often utilize disulfide bonds as redox sensors for adaptive regulation of channel conformations in response to diverse physiological environments. In this study, we developed novel synthetic ion channels capable of reversibly switching their ion-transport capabilities by incorporating multiple disulfide bonds into artificial systems. X-ray structural analysis and electrophysiological experiments demonstrated that these disulfide-bridged molecules possess well-defined tubular cavities and can be efficiently inserted into lipid bilayers to form artificial ion channels. More importantly, the disulfide bonds in these molecules serve as redox-tunable switches to regulate the formation and disruption of ion-permeation pathways, thereby achieving a transition in the transmembrane transport process between the ON and OFF states.

The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport.

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca2+-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca2+ binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca2+ over Na+, favoring the movement of Ca2+ into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca2+ binding and thus diminishes Ca2+ permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca2+ through the channel pore.

A monomeric structure of human TMEM63A protein.

OSCA/TMEM63 is a newly identified family of mechanically activated (MA) ion channels in plants and animals, respectively, which convert physical forces into electrical signals or trigger intracellular cascades and are essential for eukaryotic physiology. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. However, the molecular architecture of the mammalian TMEM63 proteins remains unclear. Here we elucidate the structure of human TMEM63A in the presence of calcium by single particle cryo-EM, revealing a distinct monomeric architecture containing eleven transmembrane helices. It has structural similarity to the single subunit of the Arabidopsis thaliana OSCA proteins. We locate the ion permeation pathway within the monomeric configuration and observe a nonprotein density resembling lipid. These results lay a foundation for understanding the structural organization of OSCA/TMEM63A family proteins.

Synthetic cation channel: reconstructing the ion permeation pathway of TRPA1 in an artificial system.

A novel artificial cation channel was developed by rebuilding the ion permeation pathway of the natural channel protein (TRPA1) in a synthetic system. This tubular molecule can effectively embed into lipid bilayers and form transmembrane channels, thereby mediating cation transport. Furthermore, due to its carboxyl-modified ion permeation pathway, the transport activity of this artificial channel can be modulated by the pH of the buffer solution.

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.

Mechanism underlying delayed rectifying in human voltage-mediated activation Eag2 channel

The transmembrane voltage gradient is a general physico-chemical cue that regulates diverse biological function through voltage-gated ion channels. How voltage sensing mediates ion flows remains unknown at the molecular level. Here, we report six conformations of the human Eag2 (hEag2) ranging from closed, pre-open, open, and pore dilation but non-conducting states captured by cryo-electron microscopy (cryo-EM). These multiple states illuminate dynamics of the selectivity filter and ion permeation pathway with delayed rectifier properties and Cole-Moore effect at the atomic level. Mechanistically, a short S4-S5 linker is coupled with the constrict sites to mediate voltage transducing in a non-domain-swapped configuration, resulting transitions for constrict sites of F464 and Q472 from gating to open state stabilizing for voltage energy transduction. Meanwhile, an additional potassium ion occupied at positions S6 confers the delayed rectifier property and Cole-Moore effects. These results provide insight into voltage transducing and potassium current across membrane, and shed light on the long-sought Cole-Moore effects.

Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization.

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Exploring the gating mechanisms of the TRPM3 channel.

TRP channels are polymodal sensors that detect a variety of physiological stimuli. TRPM3, a member of the TRP channel family, is a nonselective cation channel implicated in disorders throughout the body, including in diabetes, neurodevelopmental disorders, chronic inflammation, and chronic fatigue syndrome. As such, understanding TRPM3 function and dysfunction could provide valuable insight into physiological functioning and treatment. While TRPM3 has many isoforms, the isoform studied here is the most well-characterized TRPM3 isoform, TRPM3α2. TRPM3α2 is sensitive to heat, depolarizing membrane voltages, and ligands such as pregnenolone sulfate (PS). While PS is not specific for TRPM3, it is currently the most used agonist for the channel as it activates the channel at physiological concentrations and elicits an intracellular signaling cascade typical of TRPM3 stimulation. TRPM3α2 is also interesting from a mechanistic perspective because it has been proposed to have two ion permeation pathways, one at the central axis within the pore domain formed by S5-S6 helices and activated by PS, and a second activated by other agonists and located within S1-S4 domains that resemble voltage-sensing domains in voltage-activated potassium (Kv) channels. We began by identifying ionic conditions where we could measure macroscopic ionic currents during test depolarizations and tail currents upon membrane repolarization. We find that symmetrical CsCl based solutions seem to stabilize the open state of TRPM3, whether examining voltage-dependent activation at room temperature in the absence of other activators, or in the presence of PS, enabling robust resolution of tail current. Using tail current measurements, we observed that the conductance (G) - voltage (V) relationship for TRPM3 is complex, with distinct components at negative and positive membrane voltages. We are currently exploring ligands thought to activate the alternate permeation pathway and using mutagenesis to localize the PS binding site.

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. Each P2X receptor channel subunit consists of two transmembrane helices, a large extracellular domain that contains the ATP binding sites at subunit interfaces, and intracellular N-and C-termini. P2X1-4 and P2X7 receptor channels are cation permeable, whereas P2X5 has been reported to have a significant anion permeability. 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 at the intracellular end of the pore. We identified 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 and that these structural elements play a critical role in determining the ion selectivity of P2X receptor channels.

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) in Cav1.1 and Nav1.4. Vigorous structural and functional studies suggest that mutations of the arginine residues of VSDs destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of an aberrant ion permeation pathway, the gating pore. Presently, non-physiological leak currents through the gating pore, known as the gating pore currents, are thought to underlie HypoPP. In this study, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium ion channel (mKir2.1) and HypoPP2-associated Nav1.4 missense variants, p.R669H, p.R672H, p.R1135H or p.R669H/p.R672H (double mutations, DMT). Whole-cell patch clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane electrical potential of the cells to −90 mV or below (comparable to myofibers), and that p.R669H and DMT induced notable proton-based gating pore currents. Using a ratiometric pH indicator, SNARF-4F, we observed that the proton transports at higher rate in p.R669H and DMT compared to wild-type (WT) control. The expression of the HypoPP2-associated Nav1.4 variants was similar to the WT level based on our newly developed membrane protein targeting assay, indicating that the higher proton transport rates found in our fluorometric measurements for p.R669H and DMT are indeed ascribed to the gating pore currents. Our optical method has a potential to serve as an in vitro platform for rapidly characterizing variants associated with HypoPP and other channelopathies, and for subsequent high-throughput drug screen. Support: JP19ek0109230 and JP22bm0804005(AMED), JP18K07524 and JP22K07493(JSPS), Takeda Science Foundation, Mochida Memorial Foundation, and DC01782(NIH).

Membrane-Active Molecular Machines.

Both biological and artificial membrane transporters mediate passive transmembrane ion flux predominantly via either channel or carrier mechanisms, tightly regulating the transport of materials entering and exiting the cell. One early elegant example unclassifiable as carriers or channels was reported by Smith who derivatized a phospholipid molecule into an anion transporter, facilitating membrane transport via a two-station relay mechanism (Smith et al. J. Am. Chem. Soc. 2008, 130, 17274-17275). Our journey toward blurring or even breaking the boundaries defined by the carrier and channel mechanisms starts in January of 2018 when seeing a child swinging on the swing at the playground park. Since then, I have been wondering whether we could build a nanoscale-sized molecular swing able to perform the swing function at the molecular level to induce transmembrane ion flux. Such research journey culminates in several membrane-active artificial molecular machines, including molecular swings, ion fishers, ion swimmers, rotors, tetrapuses and dodecapuses that permeabilize the membrane via swinging, ion-fishing, swimming, rotating, or swing-relaying actions, respectively. Except for molecular ion swimmers, these unconventional membrane transporters in their most stable states readily span across the entire membrane in a way akin to channels. With built-in flexible arms that can swing or bend in the dynamic membrane environment, they transport ions via constantly changing ion permeation pathways that are more defined than carriers but less defined than channels. Applying the same benzo-crown ether groups as the sole ion-binding and -transporting units, these transporters however differ immensely in ion transport property. While the maximal K+ transport activity is achieved by the molecular swing also termed "motional channel" that displays an EC50 value of 0.021 mol % relative to lipid and transports K+ ions at rate 27% faster than gramicidin A, the highest K+/Na+ selectivity of 18.3 is attained by the molecular ion fisher, with the highest Na+/K+ selectivity of 13.7 by the molecular dodecapus. Having EC50 values of 0.49-1.60 mol % and K+/Na+ values of 1.1-6.3, molecular rotors and tetrapuses are found to be generally active but weakly to moderately K+-selective. For molecular ion swimmers that contain 10 to 14 carbon atom alkyl linkers, they all turn out to be highly active (EC50 = 0.18-0.41 mol %) and highly selective (RK+/RNa+ = 7.0-9.5) transporters. Of special note are crown ether-appended molecular dodecapuses that establish the C60-fullerene core as an excellent platform to allow for a direct translation of solution binding affinity to transmembrane ion transport selectivity, providing a de novo basis for rationally designing artificial ion transporters with high transport selectivity. Considering remarkable cytotoxic activities displayed by molecular swings and ion swimmers, the varied types of existing and emerging unconventional membrane transporters with enhanced activities and selectivities eventually might lead to medical benefits in the future.

Role of cytoplasmic cap in determining the ion selectivity of P2X receptor channels

Trimeric P2X receptors are ATP-activated ion channels, with each subunit consisting of an intracellular N- and C- termini, a large extracellular domain forming ATP binding sites, and two transmembrane helices (TMs) that form an ion permeation pathway. In recent structures of human P2X3 receptors, a cytoplasmic cap formed by the N- and C- termini covers the central ion permeation pathway, leaving only small lateral fenestrations near or within the membrane for ions to enter or exit the pore. To investigate whether these fenestrations play a role in determining the ion selectivity of P2X receptors, we identified an acidic residue (E17 in rP2X2) that lines the lateral fenestrations and is conserved in subtypes that are cation selective but replaced by a basic residue in P2X5 receptors (K17 in mP2X5), a subtype reported to have significant chloride permeability.

Explanation of Structure and Function of kv1.3 Potent Blocker from Mesobuthus eupeus Venom Gland: A New Promise in Drug Development

Background: Scorpions and other venomous animals are sought with great concern because venom is a source of novel peptides with exciting features. Some toxins of scorpion venom are effectors of potassium channels. Previous studies strongly support the importance of potassium channel toxins for use as pharmacological tools or potential drugs. Objectives: Here, a three-dimensional (3-D) structure and function of a potent acidic blocker of the human voltage-gated potassium ion channel, Kv1.3, previously identified in the scorpion Mesobuthus eupeus venom gland, were interpreted. Methods: The 3-D structure of meuK2-2 was generated using homology modeling. The interaction of meuK2-2 with the Kv1.3 channel was evaluated using a computational protocol employing peptide-protein docking experiments, pose clustering, and 100 ns molecular dynamic simulations to make the 3-D models of the meuK2-2/Kv1.3 complex trustworthy. Results: A CSα/β (cysteine-stabilized α-helical and β-sheet) fold was found for the 3-D structure of meuK2-2. In a different mechanism from what was identified so far, meuK2-2 binds to both turret and pore loop of Kv1.3 through two key residues (Ala28 and Ser11) and H-bonds. The binding of meuK2-2 induces some conformational changes to Kv1.3. Eventually, the side chain of a positively charged amino acid (His9) occupies the channel's pore. All together blocks the ion permeation pathway. Conclusions: MeuK2-2 could block Kv1.3 by a new mechanism. So, it could be a unique target for further investigations to develop a pharmacological tool and potential drug.

A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel.

G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+ , while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+ , suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+ -selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q-sensitive K+ current and the second a TPN-Q-resistant Li+ current. The results show that a novel Li+ -permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix, S148F and T151A also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations. KEY POINTS: G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important roles in controlling excitation of cells in various organs, such as the brain and the heart. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited mutants of Kir3.2 and Kir3.4. Here we show that a novel Na+ , Li+ -permeable and blocker-resistant pathway exists in an inherited mutant, Kir3.2 G156S, in addition to the conventional ion conducting pathway formed by the selectivity filter (SF). Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.