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Background: Transient receptor potential vanilloid subfamily member 1 (TRPV1) channels are important integrators of noxious stimuli with pronounced expression in nociceptive neurons. The experimental local anesthetic, QX-314, a quaternary (i.e., permanently charged) lidocaine derivative, recently has been shown to interact with and permeate these channels to produce nociceptive and sensory blockade in animals in vivo. However, little is known about the specific interactions between QX-314 and TRPV1 channels. Thus, the authors examined the mechanistic basis by which QX-314 acts on TRPV1 channels.Methods: The authors conducted an in vitro laboratory study in which they expressed TRPV1 and TRPV4 channels in Xenopus laevis oocytes and recorded cation currents with the two-electrode voltage clamp method. They used confocal microscopy for Ca²⁺ imaging in TRPV1 transient transfected tsA201 cells. Drugs were bath-applied by gravity perfusion. Statistical analyses were performed using Student t test, ANOVA, and post tests as appropriate (P < 0.05).Results: QX-314 activated TRPV1 channels at 10, 30, and 60 mM (0.4 ± 0.1%, 3.5 ± 1.3%, and 21.5 ± 6.9% of normalized peak activation, respectively; mean ± SEM; n = 12) but not TRPV4 channels (P < 0.001). Activation by QX-314 was blocked by the TRPV1 antagonist, capsazepine (100 μM). QX-314 (60 mM) activation and blockade by capsazepine was also demonstrated in Ca²⁺ imaging studies on TRPV1-expressing tsA201 cells. At subactivating concentrations (less than 1 mM), QX-314 potently inhibited capsaicin-evoked TRPV1 currents with an IC₅₀ of 8.0 ± 0.6 μM.Conclusions: The results of this study show that the quaternary lidocaine derivative QX-314 exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations. These findings provide novel insights into the interactions between QX-314 and TRPV1 and may provide an explanation for the irritant properties of intrathecal QX-314 in mice in vivo. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization, which terminates activation; steady-state desensitization, which occurs at sub-activating proton concentrations and limits subsequent activation; and tachyphylaxis, which results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild type, but introducing an N414K mutation in the β11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Although the extracellular domain of this mutant remained sensitive to pH and underwent pH-dependent conformational changes, these conformational changes did not necessarily lead to desensitization. N414K-containing channels also remained sensitive to a known peptide modulator that increases steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to our understanding of the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the extracellular domain and the pore during channel activation and desensitization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The sodium (Na + ) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (Na V s and Ca V s, respectively). Unlike Na V s and Ca V s, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of Na V , Ca V , and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties., Competing Interests: Competing interests statement:The authors declare no competing interest.

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC., Materials and Methods: Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca 2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay., Results: Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca 2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca 2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1β, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects., Conclusion: In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers., (© 2024. The Author(s).)

Background and Purpose: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains., Experimental Approach: Here, we examine the impact of P2X2 receptor inter-subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches., Key Results: We demonstrate that predicted extracellular domain inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity., Conclusion and Implications: We provide the first structural mapping of the mutational distribution across the human population in a ligand-gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand-gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Unlike classical voltage-gated sodium (Na V ) channels, Na X has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na + )-activated channel involved in regulating Na + homeostasis. However, Na X remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human Na X channel in complex with the auxiliary β3-subunit. Na X reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na + -sensor or any evidence for a Na + -based activation mechanism in Na X . Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active Na X -QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical Na V channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the Na V channel scaffold, reshape our understanding of Na X physiology, and provide a template to demystify recalcitrant ion channels., (© 2022. The Author(s).)

Depolarizing sodium (Na + ) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity 1-8 . NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood 3,7,9-12 . NALCN, UNC79 and UNC80 are essential in rodents 2,9,13 , and mutations in human NALCN and UNC80 cause severe developmental and neurological disease 14,15 . Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition, remain unknown. Here, we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveals that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications., Competing Interests: SH, CB, JC No competing interests declared, SP Reviewing editor, eLife, (© 2022, Heusser et al.)

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on Na V 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca 2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Tissue acidification is associated with a variety of disease states, and acid-sensing ion channels (ASICs) that can sense changes in pH have gained traction as possible pharmaceutical targets. An array of modulators, ranging from small molecules to large biopharmaceuticals, are known to inhibit ASICs. Here, we summarize recent insights from animal studies to assess the therapeutic potential of ASICs in disorders such as ischemic stroke, various pain-related processes, anxiety, and cardiac pathologies. We also review the factors that present a challenge in the pharmacological targeting of ASICs, and which need to be taken into careful consideration when developing potent and selective modulators in the future., Competing Interests: Declaration of interests No interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels., (© 2021 Sheikh et al.)

Although several ionic mechanisms are known to control rate and regularity of the slow pacemaker in dopamine (DA) neurons, the core mechanism of pacing is controversial. Here we tested the hypothesis that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidinium (XG), an established blocker of gating pore currents, selectively inhibits pacemaking of DA neurons. The compound inhibited all slow pacemaking DA neurons that were tested, both in the substantia nigra pars compacta, and in the ventral tegmental area. Interestingly, bursting behavior was not affected by XG. Furthermore, the drug did not affect fast pacemaking of GABAergic neurons from substantia nigra pars reticulata neurons or slow pacemaking of noradrenergic neurons. In DA neurons, current-clamp analysis revealed that XG did not appear to affect ion channels involved in the action potential. Its inhibitory effect persisted during blockade of all ion channels previously suggested to contribute to pacemaking. RNA sequencing and voltage-clamp recordings yielded no evidence for a gating pore current to underlie the conductance. However, we could isolate a small subthreshold XG-sensitive current, which was carried by both Na + and Cl - ions. Although the molecular target of XG remains to be defined, these observations represent a step towards understanding pacemaking in DA neurons., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Søren Friis is a full-time employee of Nanion Technologies. The other authors declare no competing interests.

The voltage-gated sodium channel Na v 1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Na v 1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Na v 1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Na v 1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Na v 1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Na v 1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Persistently depolarizing sodium (Na + ) leak currents enhance electrical excitability 1,2 . The ion channel responsible for the major background Na + conductance in neurons is the Na + leak channel, non-selective (NALCN) 3,4 . NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm 4-10 . NALCN activity is under tight regulation 11-14 and mutations in NALCN cause severe neurological disorders and early death 15,16 . NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.

Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be applied to. Their application in both heterologous and native cell systems will no doubt play an increasingly important role in ion channel research., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, Na V 1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80, and FAM155A. The resulting NALCN channel complex is constitutively active and conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca 2+ . Despite the smaller number of voltage-sensing residues in NALCN, the constitutive activity is modulated by voltage, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points toward formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics., Competing Interests: The authors declare no competing interest.

Despite the sequence homology between acid-sensing ion channels (ASICs) and epithelial sodium channel (ENaCs), these channel families display very different functional characteristics. Whereas ASICs are gated by protons and show a relatively low degree of selectivity for sodium over potassium, ENaCs are constitutively active and display a remarkably high degree of sodium selectivity. To decipher if some of the functional diversity originates from differences within the transmembrane helices (M1 and M2) of both channel families, we turned to a combination of computational and functional interrogations, using statistical coupling analysis and mutational studies on mouse ASIC1a. The coupling analysis suggests that the relative position of M1 and M2 in the upper part of the pore domain is likely to remain constant during the ASIC gating cycle, whereas they may undergo relative movements in the lower part. Interestingly, our data suggest that to account for coupled residue pairs being in close structural proximity, both domain-swapped and nondomain-swapped ASIC M2 conformations need to be considered. Such conformational flexibility is consistent with structural work, which suggested that the lower part of M2 can adopt both domain-swapped and nondomain-swapped conformations. Overall, mutations to residues in the middle and lower pore were more likely to affect gating and/or ion selectivity than those in the upper pore. Indeed, disrupting the putative interaction between a highly conserved Trp/Glu residue pair in the lower pore is detrimental to gating and selectivity, although this interaction might occur in both domain-swapped and nonswapped conformations. Finally, our results suggest that the greater number of larger, aromatic side chains in the ENaC M2 helix may contribute to the constitutive activity of these channels at a resting pH. Together, the data highlight differences in the transmembrane domains of these closely related ion channels that may help explain some of their distinct functional properties., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7') and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18') residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18' residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7' side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7'A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7' and upper charges did not lead to large changes in selectivity, suggesting a different role for L7' in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18' in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional., (© 2020 Lynagh et al.)

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists., (© 2019 Gasparri et al.)

Pentameric ligand-gated ion channels (pLGICs, also known as Cys-loop receptors) are a large family of ion channels expressed in all Bilateria and in several groups of bacteria and archaea. They are activated by small-molecule neurotransmitters to mediate fast transmission at many central and peripheral nervous system synapses and are the target of several drugs and insecticides. Here we review recent advances in the field, focussing on new insights on the structure of the agonist-binding site and on newly discovered protein-protein interactions involving pLGICs.

All proteins contain characteristic backbones formed of consecutive amide bonds, which can engage in hydrogen bonds. However, the importance of these is not easily addressed by conventional technologies that only allow for side-chain substitutions. By contrast, technologies such as nonsense suppression mutagenesis and protein ligation allow for manipulation of the protein backbone. In particular, replacing the backbone amide groups with ester groups, that is, amide-to-ester mutations, is a powerful tool to examine backbone-mediated hydrogen bonds. In this minireview, we showcase examples of how amide-to-ester mutations can be used to uncover pivotal roles of backbone-mediated hydrogen bonds in protein recognition, folding, function, and structure., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers., (© 2018 Wang et al.)

Retigabine is an antiepileptic drug and the first voltage-gated potassium (Kv) channel opener to be approved for human therapeutic use. Retigabine is thought to interact with a conserved Trp side chain in the pore of KCNQ2-5 (Kv7.2-7.5) channels, causing a pronounced hyperpolarizing shift in the voltage dependence of activation. In this study, we investigate the functional stoichiometry of retigabine actions by manipulating the number of retigabine-sensitive subunits in concatenated KCNQ3 channel tetramers. We demonstrate that intermediate retigabine concentrations cause channels to exhibit biphasic conductance-voltage relationships rather than progressive concentration-dependent shifts. This suggests that retigabine can exert its effects in a nearly "all-or-none" manner, such that channels exhibit either fully shifted or unshifted behavior. Supporting this notion, concatenated channels containing only a single retigabine-sensitive subunit exhibit a nearly maximal retigabine effect. Also, rapid solution exchange experiments reveal delayed kinetics during channel closure, as retigabine dissociates from channels with multiple drug-sensitive subunits. Collectively, these data suggest that a single retigabine-sensitive subunit can generate a large shift of the KCNQ3 conductance-voltage relationship. In a companion study (Wang et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812014), we contrast these findings with the stoichiometry of a voltage sensor-targeted KCNQ channel opener (ICA-069673), which requires four drug-sensitive subunits for maximal effect., (© 2018 Yau et al.)

Acid-sensing ion channels (ASICs) are proton-gated ion channels broadly expressed in the vertebrate nervous system, converting decreased extracellular pH into excitatory sodium current. ASICs were previously thought to be a vertebrate-specific branch of the DEG/ENaC family, a broadly conserved but functionally diverse family of channels. Here, we provide phylogenetic and experimental evidence that ASICs are conserved throughout deuterostome animals, showing that ASICs evolved over 600 million years ago. We also provide evidence of ASIC expression in the central nervous system of the tunicate, Oikopleura dioica Furthermore, by comparing broadly related ASICs, we identify key molecular determinants of proton sensitivity and establish that proton sensitivity of the ASIC4 isoform was lost in the mammalian lineage. Taken together, these results suggest that contributions of ASICs to neuronal function may also be conserved broadly in numerous animal phyla., Competing Interests: The authors declare no conflict of interest.

Objective: Unexplained cardiac arrest (UCA) is often the first manifestation of an inherited arrhythmogenic disease. Genetic testing in UCA is challenging due to the complexities of variant interpretation in the absence of supporting cardiac phenotype. We aimed to investigate if a KCNQ1 variant [p.(Pro64&#95;Pro70del)], previously reported as pathogenic, contributes to the long-QT syndrome phenotype, co-segregates with disease or affects KCNQ1 function in vitro., Methods: DNA was extracted from peripheral blood of a 22-year-old male after resuscitation from UCA. Targeted exome sequencing was performed using the TruSight-One Sequencing Panel (Illumina). Variants in 190 clinically relevant cardiac genes with minor allele frequency < 1% were analyzed according to the guidelines of the American College of Medical Genetics. Functional characterization was performed using site-directed mutagenesis, expression in Xenopus laevis oocytes using the two-electrode voltage-clamp technique., Results: The 12-lead ECG, transthoracic echocardiography and coronary angiography after resuscitation showed no specific abnormalities. Two variants were identified: c.190&#95;210del in-frame deletion in KCNQ1 (p.Pro64&#95;Pro70del), reported previously as pathogenic and c.2431C > A in PKP2 (p.Arg811Ser), classified as likely benign. Two asymptomatic family members with no evident phenotype hosted the KCNQ1 variant. Functional studies showed that the wild-type and mutant channels have no significant differences in current levels, conductance-voltage relationships, as well as activation and deactivation kinetics, in the absence and presence of the auxiliary subunit KCNE1., Conclusions: Based on our data and previous reports, available evidence is insufficient to consider the variant KCNQ1:c.190&#95;210del as pathogenic. Our findings call for cautious interpretation of genetic tests in UCA in the absence of a clinical phenotype.

Glutamate-gated chloride channels (GluCls) are neurotransmitter receptors that mediate crucial inhibitory signaling in invertebrate neuromuscular systems. Their role in invertebrate physiology and their absence from vertebrates make GluCls a prime target for antiparasitic drugs. GluCls from flatworm parasites are substantially different from and are much less understood than those from roundworm and insect parasites, hindering the development of potential therapeutics targeting GluCls in flatworm-related diseases such as schistosomiasis. Here, we sought to dissect the molecular and chemical basis for ligand recognition in the extracellular glutamate binding site of SmGluCl-2 from Schistosoma mansoni, using site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings. Our results indicate that aromatic residues in ligand binding loops A, B, and C are important for SmGluCl-2 function. Loop C, which differs in length compared to other pentameric ligand-gated ion channels (pLGICs), contributes to ligand recognition through both an aromatic residue and two vicinal threonine residues. We also show that, in contrast to other pLGICs, the hydrophobic channel gate in SmGluCl-2 extends from the 9' position to the 6' position in the channel-forming M2 helix. The 6' and 9' positions also seem to control sensitivity to the pore blocker picrotoxin.

Fluorescent labels offer the capability to follow conformational dynamics of membrane proteins, but signal detection in such recordings is inherently difficult to achieve in a cell membrane and lacks sufficient time resolution to follow physiologically relevant transitions. Here, we develop high-sensitivity patch-clamp fluorometry (hsPCF), a fluorescence-based approach that results in up to 10-fold increased signals and affords 50-fold faster fluorescence recordings than previous methods. The increased time resolution is paired with a very high versatility in terms of the choice of fluorescent dye, cell type, and protein of interest. We highlight this versatility by providing insight into the conformational dynamics of both ligand- and voltage-gated ion channels using fluorescent labels introduced in extracellular or transmembrane positions while changing either the extra- or intracellular solutions. Together, hsPCF will thus enable the future study of membrane-embedded proteins with sufficient temporal resolution to resolve conformational dynamics., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2-5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD-pore coupling via PIP2, and thereby influences the unique gating effects of retigabine., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

A growing body of evidence links certain aspects of nonsteroidal anti-inflammatory drug (NSAID) pharmacology with acid-sensing ion channels (ASICs), a small family of excitatory neurotransmitter receptors implicated in pain and neuroinflammation. The molecular basis of NSAID inhibition of ASICs has remained unknown, hindering the exploration of this line of therapy. Here, we characterized the mechanism of inhibition, explored the molecular determinants of sensitivity, and sought to establish informative structure-activity relationships, using electrophysiology, site-directed mutagenesis, and voltage-clamp fluorometry. Our results show that ibuprofen is an allosteric inhibitor of ASIC1a, which binds to a crucial site in the agonist transduction pathway and causes conformational changes that oppose channel activation. Ibuprofen inhibits several ASIC subtypes, but certain ibuprofen derivatives show some selectivity for ASIC1a over ASIC2a and vice versa. These results thus define the NSAID/ASIC interaction and pave the way for small-molecule drug design targeting pain and inflammation.

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the "GAS belt" in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cys-loop receptors mediate fast synaptic transmission in the nervous system, and their dysfunction is associated with a number of diseases. While some sequence variability is essential to ensure specific recognition of a chemically diverse set of ligands, other parts of the underlying amino acid sequences show a high degree of conservation, possibly to preserve the overall structural fold across the protein family. In this study, we focus on the only two absolutely conserved residues across the Cys-loop receptor family, two Trp side chains in the WXD motif of Loop D and in the WXPD motif of Loop A. Using a combination of conventional mutagenesis, unnatural amino acid incorporation, immunohistochemistry and MD simulations, we demonstrate the crucial contributions of these two Trp residues to receptor expression and function in two prototypical Cys-loop receptors, the anion-selective GlyR α1 and the cation-selective nAChR α7. Specifically, our results rule out possible electrostatic contributions of these Trp side chains and instead suggest that the overall size and shape of this aromatic pair is required in stabilizing the Cys-loop receptor extracellular domain.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Ion channels are membrane-spanning proteins that control the flow of ions across biological membranes through an aqueous pathway. The opening or closing of this pore can be controlled by a myriad of physiological inputs (voltage, ligands, temperature, metabolites, pH), which in turn allow for the controlled flux of ions across membranes, resulting in the generation of minute electrical signals. The functional implications of ion channel function on physiological processes are vast. Electrical impulses, in the form of action potentials or diverse chemo-electrical signals, coordinate the syncytium of the heart beat, support a myriad of neuronal communication pathways, insulin secretion, and are central to the immune response, with more roles being discovered virtually everyday. Thus, ion channel function is a biophysical process that is central to biological life at many levels. And with over 500 channel-forming subunits known today in humans, this large class of proteins is also increasingly recognised as important drug targets, as inherited or acquired ion channel dysfunction are known causes of disease.

In this issue of Structure, Ulens and colleagues demonstrate how an elegant combination of complementary functional and structural approaches can uncover both binding sites and conformational consequences associated with the Alzheimer's drug memantine binding to an ion channel., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.

Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term "chemoreceptor" emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands.

Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445-Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001.

Voltage-gated potassium channels elicit membrane hyperpolarization through voltage-sensor domains that regulate the conductive status of the pore domain. To better understand the inherent basis for the open-closed equilibrium in these channels, we undertook an atomistic scan using synthetic fluorinated derivatives of aromatic residues previously implicated in the gating of Shaker potassium channels. Here we show that stepwise dispersion of the negative electrostatic surface potential of only one site, Phe481, stabilizes the channel open state. Furthermore, these data suggest that this apparent stabilization is the consequence of the amelioration of an inherently repulsive open-state interaction between the partial negative charge on the face of Phe481 and a highly co-evolved acidic side chain, Glu395, and this interaction is potentially modulated through the Tyr485 hydroxyl. We propose that the intrinsic open-state destabilization via aromatic repulsion represents a new mechanism by which ion channels, and likely other proteins, fine-tune conformational equilibria.