Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

Receptors on Microglia.

Microglial cells are the most receptive cells in the central nervous system (CNS), expressing several classes of receptors reflecting their immune heritage and newly acquired neural specialisation. Microglia possess, depending on the particular context, receptors to neurotransmitters and neuromodulators as well as immunocompetent receptors. This rich complement allows microglial cells to monitor the functional status of the nervous system, contribute actively to the regulation of neural activity and plasticity and homeostasis, and guard against pathogens as well as other challenges to the CNS's integrity and function.

Confinement controls the directional cell responses to fluid forces.

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly-but not moderately-confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor.

Serum and glucocorticoid-regulated kinase 1 (SGK1) as an emerging therapeutic target for cardiac diseases.

Cardiac diseases encompass a wide range of conditions that affect the structure and function of the heart. These conditions are a leading cause of morbidity and mortality worldwide. The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase that plays a significant role in various cellular processes, including cell survival and stress response. Alterations in SGK1 activity can have significant impacts on health and disease. Multiple research findings have indicated that SGK1 is associated with heart disease due to its involvement in cardiac hypertrophy and fibrosis. This article reviews different signaling pathways associated with SGK1 activity in various heart conditions, including the SGK1/NF-κB and PI3K/SGK1 pathways.

The Voltage-Gated Calcium Channel α2δ Subunit in Neuropathic Pain.

Neuropathic pain (NP) is a chronic pain caused by injury or disease of the somatosensory nervous system, or it can be directly caused by disease. It often presents with clinical features like spontaneous pain, hyperalgesia, and dysesthesia. At present, voltage-gated calcium ion channels (VGCCs) are known to be closely related to the development of NP, especially the α2δ subunit. The α2δ subunit is a regulatory subunit of VGCCs. It exists mainly in the brain and peripheral nervous system, especially in nerve cells, and it plays a crucial part in regulating presynaptic and postsynaptic functions. Furthermore, the α2δ subunit influences neuronal excitation and pain signaling by promoting its expression and localization through binding to VGCC-related subunits. The α2δ subunit is widely used in the management of NP as a target of antiepileptic drugs gabapentin and pregabalin. Although drug therapy is one of the treatments for NP, its clinical application is limited due to the adverse reactions caused by drug therapy. Therefore, further research on the therapeutic target α2δ subunit is needed, and attempts are made to obtain an effective treatment for relieving NP without side effects. This review describes the current associated knowledge on the function of the α2δ subunit in perceiving and modulating NP.

The evolution of patch-clamp electrophysiology: robotic, multiplex, and dynamic.

The patch-clamp technique has been the gold standard for analysis of excitable cells. Since its development in the 1980s it has contributed immensely to our understanding of neurons, muscle cells, and cardiomyocytes, and the ion channels and receptors that reside within them. This technique, predicated on Ohm's law, enables precise measurements of macroscopic excitability patterns, and ionic and gating conductances that can be assessed even down to the single channel level. Over the years, patch-clamp electrophysiology has undergone extensive modifications, with the introduction of new applications that have enhanced its power and reach. The most recent evolution of this technique occurred with the introduction of robotic high throughput automated platforms that enable high quality simultaneous recordings, in both voltage- and current-clamp modes, from 10s to 100s of cells, including cells freshly isolated from their native tissues. Combined with new dynamic-clamp applications, these new methods provide increasingly powerful tools for studying the contributions of ion channels and receptors to electrogenesis. In this brief review, we provide an overview of these enhanced patch-clamp techniques, followed by some of the applications presently being pursued, and a perspective into the potential future of the patch-clamp method. Significance Statement The patch-clamp technique, introduced in the 1980s, has revolutionized understanding of electrogenesis. Predicated on Ohm's law, this approach facilitates exploration of ionic conductances, gating mechanisms of ion channels and receptors, and their roles in neuronal, muscular, and cardiac excitability. Robotic platforms for high-throughput patch-clamp, and dynamic-clamp, have recently expanded its reach. Here, we outline new advances in patch-clamp including high throughput analysis of freshly-isolated neurons, and discuss the increasingly powerful trajectory of new patch-clamp techniques.

Voltage-gated ion channel's gene expression in the myocardium of embryo and adult chickens.

The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C, KCNH6, RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane-clock¼ (hyperpolarization-activated current (If), Ca2+-current L-type (ICaL), Na+-current (INa) and the slow component of the delayed rectifier K+-current (IKs)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane- clock¼ and «Ca2+-clock¼.

Harnessing Deep Learning Methods for Voltage-Gated Ion Channel Drug Discovery.

Voltage-gated ion channels (VGICs) are pivotal in regulating electrical activity in excitable cells and are critical pharmaceutical targets for treating many diseases including cardiac arrhythmia and neuropathic pain. Despite their significance, challenges such as achieving target selectivity persist in VGIC drug development. Recent progress in deep learning, particularly diffusion models, has enabled the computational design of protein binders for any clinically relevant protein based solely on its structure. These developments coincide with a surge in experimental structural data for VGICs, providing a rich foundation for computational design efforts. This review explores the recent advancements in computational protein design using deep learning and diffusion methods, focusing on their application in designing protein binders to modulate VGIC activity. We discuss the potential use of these methods to computationally design protein binders targeting different regions of VGICs, including the pore domain, voltage-sensing domains, and interface with auxiliary subunits. We provide a comprehensive overview of the different design scenarios, discuss key structural considerations, and address the practical challenges in developing VGIC-targeting protein binders. By exploring these innovative computational methods, we aim to provide a framework for developing novel strategies that could significantly advance VGIC pharmacology and lead to the discovery of effective and safe therapeutics.

The role of calcium channels in osteoporosis and their therapeutic potential.

Osteoporosis, a systemic skeletal disorder marked by diminished bone mass and compromised bone microarchitecture, is becoming increasingly prevalent due to an aging population. The underlying pathophysiology of osteoporosis is attributed to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoclasts play a crucial role in the development of osteoporosis through various molecular pathways, including the RANK/RANKL/OPG signaling axis, cytokines, and integrins. Notably, the calcium signaling pathway is pivotal in regulating osteoclast activation and function, influencing bone resorption activity. Disruption in calcium signaling can lead to increased osteoclast-mediated bone resorption, contributing to the progression of osteoporosis. Emerging research indicates that calcium-permeable channels on the cellular membrane play a critical role in bone metabolism by modulating these intracellular calcium pathways. Here, we provide an overview of current literature on the regulation of plasma membrane calcium channels in relation to bone metabolism with particular emphasis on their dysregulation during the progression of osteoporosis. Targeting these calcium channels may represent a potential therapeutic strategy for treating osteoporosis.

Human pain channelopathies.

There has been significant progress in our understanding of the molecular basis by which nociceptors transduce and transmit noxious (tissue damaging) stimuli. This is dependent on ion channels, many of which are selectively expressed in nociceptors. Mutations in such proteins have recently been linked to inherited pain disorders in humans. An exemplar is the voltage-gated sodium channel (VGSC) NaV1.7. Loss of function mutations in NaV1.7 result in congenital inability to experience pain while gain-of-function mutations can cause a number of distinct neuropathic pain disorders, including erythromelalgia, paroxysmal extreme pain disorder, and small-fiber neuropathy. Furthermore, variants in the VGSCs 1.8 and 1.9 have also been linked to human pain disorders. There is a correlation between the impact of mutations on the biophysical properties of the ion channel and the severity of the clinical phenotype. Pain channelopathies are not restricted to VGSCs: a mutation in the ligand-gated ion channel TRPA1, (which responds to environmental irritants) causes a familial episodic pain disorder. Ion channel variants have also been linked to more common neuropathic pain disorders such as painful diabetic neuropathy. Not only do these ion channels present targets for novel analgesics, but stratification based on genotype may improve treatment selection of existing analgesics.

Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na+ restriction.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

Protein nanoparticles induce the activation of voltage-dependent non-selective ion channels to modulate biological osmotic pressure in cytotoxic cerebral edema.

Introduction: Cytotoxic cerebral edema is a serious complication associated with cerebral ischemic stroke and is widely treated using the hypertonic dehydrant. Here, we propose, for the first time, the decrease of intracellular osmosis as a treatment strategy for alleviating cytotoxic cerebral edema. Methods: We established a fluorescence resonance energy transfer-based intermediate filament tension probe for the study and in situ evaluation of osmotic gradients, which were examined in real-time in living cells from primary cultures as well as cell lines. The MCAO rat model was used to confirm our therapy of cerebral edema. Results: Depolymerization of microfilaments/microtubules and the production of NLRP3 inflammasome resulted in an abundance of protein nanoparticles (PNs) in the glutamate-induced swelling of astrocytes. PNs induced changes in membrane potential and intracellular second messengers, thereby contributing to hyper-osmosis and the resultant astrocyte swelling via the activation of voltage-dependent nonselective ion channels. Therefore, multiple inhibitors of PNs, sodium and chloride ion channels were screened as compound combinations, based on a decrease in cell osmosis and astrocyte swelling, which was followed by further confirmation of the effectiveness of the compound combination against alleviated cerebral edema after ischemia. Discussion: The present study proposes new pathological mechanisms underlying "electrophysiology-biochemical signal-osmotic tension," which are responsible for cascade regulation in cerebral edema. It also explores various compound combinations as a potential treatment strategy for cerebral edema, which act by multi-targeting intracellular PNs and voltage-dependent nonselective ion flux to reduce astrocyte osmosis.

Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Obesity Arrhythmias: Role of IL-6 Trans-Signaling.

Obesity is a chronic disease that is rapidly increasing in prevalence and affects more than 600 million adults worldwide, and this figure is estimated to increase by at least double by 2030. In the United States, more than one-third of the adult population is either overweight or obese. The global obesity epidemic is a major risk factor for the development of life-threatening arrhythmias occurring in patients with long QT, particularly in conditions where multiple heart-rate-corrected QT-interval-prolonging mechanisms are simultaneously present. In obesity, excess dietary fat in adipose tissue stimulates the release of immunomodulatory cytokines such as interleukin (IL)-6, leading to a state of chronic inflammation in patients. Over the last decade, increasing evidence has been found to support IL-6 signaling as a powerful predictor of the severity of heart diseases and increased risk for ventricular arrhythmias. IL-6's pro-inflammatory effects are mediated via trans-signaling and may represent a novel arrhythmogenic risk factor in obese hearts. The first selective inhibitor of IL-6 trans-signaling, olamkicept, has shown encouraging results in phase II clinical studies for inflammatory bowel disease. Nevertheless, the connection between IL-6 trans-signaling and obesity-linked ventricular arrhythmias remains unexplored. Therefore, understanding how IL-6 trans-signaling elicits a cellular pro-arrhythmic phenotype and its use as an anti-arrhythmic target in a model of obesity remain unmet clinical needs.

Iterative design of polymer fabric cathode for metal-ion batteries.

Organic electrode materials (OEMs) have attracted significant attention for use in aqueous zinc-ion batteries (AZIBs) because of their abundant resources and flexible designability. However, the development of high-performance OEMs is strongly hindered by their high solubility, poor conductivity, sluggish ion diffusion kinetics, and difficult coordination toward Zn2+. Herein, inspired by fabric crafts, we have designed a robust polymer fabric through the iterative evolution of the building blocks from point to line and plane. The evolution from point to line could not only improve the structural stability and electrical conductivity but also adjust the active site arrangement to enable the storage of Zn2+. In addition to further boosting the aforementioned properties, the evolution from line to plane could also facilitate the construction of noninterference channels for ion migration. Accordingly, the poly(1,4,5,8-naphthalenetetracarboxylic dianhydride/2,3,5,6-tetraaminocyclohexa-2,5-diene-1,4-dione) (PNT) polymer fabric has the most enhanced structural stability, optimized active site arrangement, improved electrical conductivity, and suitable ion channels, resulting in a record-high capacity retention of 96% at a high mass loading of 56.9mg cm-2 and a stable cycle life of more than 20,000 cycles at 150C (1C=200 mA g-1) in AZIBs. In addition, PNT exhibits universality for a wide range of ions in organic electrolyte systems, such as Li/Na/K-ion batteries. Our iterative design of polymer fabric cathode has laid the foundation for the development of advanced OEMs to promote the performance of metal-ion batteries.

Vascular Function and Ion Channels in Alzheimer's Disease.

This review paper explores the critical role of vascular ion channels in the regulation of cerebral artery function and examines the impact of Alzheimer's disease (AD) on these processes. Vascular ion channels are fundamental in controlling vascular tone, blood flow, and endothelial function in cerebral arteries. Dysfunction of these channels can lead to impaired cerebral autoregulation, contributing to cerebrovascular pathologies. AD, characterized by the accumulation of amyloid beta (Aß) plaques and neurofibrillary tangles, has been increasingly linked to vascular abnormalities, including altered vascular ion channel activity. Here, we briefly review the role of vascular ion channels in cerebral blood flow control and neurovascular coupling. We then examine the vascular defects in AD, the current understanding of how AD pathology affects vascular ion channel function, and how these changes may lead to compromised cerebral blood flow and neurodegenerative processes. Finally, we provide future perspectives and conclusions. Understanding this topic is important as ion channels may be potential therapeutic targets for improving cerebrovascular health and mitigating AD progression.

The role of the ubiquitin system in the onset and reversal of neuropathic pain.

Neuropathic pain (NP) remains one of the world's most difficult problems, and people suffering from NP have their quality of life affected to a great extent and constantly suffer from pain. Sensitization of injurious receptors, ectopic firing of afferent nerves after nerve injury, and coupling between sympathetic and sensory neurons are involved in the onset or development of NP, but the pathogenesis of NP is still not well understood. We found that the ubiquitin system is involved in the pathogenesis of NP and has a crucial role in it. The ubiquitin system can be involved in the onset or reversal of NP by affecting ion channels, cellular signal transduction, glial cells, and the regulation of non-coding RNAs. This provides new ideas for the treatment of NP. The ubiquitin system may be a new effective target for the treatment of NP. A continued, in-depth understanding of the mechanisms of the ubiquitin system involved in NP could further refine the study of analgesic targets and improve pharmacological studies.

Ultrasound pulse repetition frequency preferentially activates different neuron populations independent of cell type.

OBJECTIVE: Transcranial ultrasound stimulation serves as an external input to a neuron, and thus the evoked response relies on neurons' intrinsic properties. Neural activity is limited to a couple hundred hertz and often exhibits preference to input frequencies. Accordingly, ultrasound pulsed at specific physiologic pulse repetition frequencies (PRFs) may selectively engage neurons with the corresponding input frequency preference. However, most ultrasound parametric studies examine the effects of supraphysiologic PRFs. It remains unclear whether pulsing ultrasound at different physiologic PRFs could activate distinct neurons in the awake mammalian brain. Approach: We recorded cellular calcium responses of individual motor cortex neurons to ultrasound pulsed at PRFs of 10, 40, and 140 Hz in awake mice. We compared the evoked responses across these PRFs in the same neurons. To further understand the cell-type dependent effects, we categorized the recorded neurons as parvalbumin positive fast spiking interneurons or putative excitatory neurons and analyzed single-cell mechanosensitive channel expression in mice and humans using the Allen Brain Institute's RNA-sequencing databases. Main results: We discovered that many neurons were preferentially activated by only one PRF and different PRFs selectively engaged distinct neuronal populations. Ultrasound-evoked cellular calcium responses exhibited the same characteristics as those naturally occurring during spiking, suggesting that ultrasound increases intrinsic neuronal activity. Furthermore, evoked responses were similar between fast-spiking inhibitory neurons and putative excitatory neurons. Thus, variation in individual neuron's cellular properties dominates ultrasound-evoked response heterogeneity, consistent with our observed cell-type independent expression patterns of mechanosensitive channels across individual neurons in mice and humans. Finally, ultrasound transiently increased network synchrony without producing prolonged over-synchronization that could be detrimental to neural circuit functions. Significance: These results highlight the feasibility of activating distinct neuronal subgroups by varying PRF and the potential to improve neuromodulation effects by combining physiologic PRFs. .

Mechanosensitive ion channels in glaucoma pathophysiology.

Force sensing is a fundamental ability that allows cells and organisms to interact with their physical environment. The eye is constantly subjected to mechanical forces such as blinking and eye movements. Furthermore, elevated intraocular pressure (IOP) can cause mechanical strain at the optic nerve head, resulting in retinal ganglion cell death (RGC) in glaucoma. How mechanical stimuli are sensed and affect cellular physiology in the eye is unclear. Recent studies have shown that mechanosensitive ion channels are expressed in many ocular tissues relevant to glaucoma and may influence IOP regulation and RGC survival. Furthermore, variants in mechanosensitive ion channel genes may be associated with risk for primary open angle glaucoma. These findings suggest that mechanosensitive channels may be important mechanosensors mediating cellular responses to pressure signals in the eye. In this review, we focus on mechanosensitive ion channels from three major channel families-PIEZO, two-pore potassium and transient receptor potential channels. We review the key properties of these channels, their effects on cell function and physiology, and discuss their possible roles in glaucoma pathophysiology.

Proline substitutions in the ASIC1 ß11-12 linker slow desensitization.

Desensitization is a prominent feature of nearly all ligand gated ion channels. Acid-sensing ion channels (ASIC) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or "flipping" of a short linker joining the 11th and 12th beta sheets in the extracellular domain. In the resting and active states this ß11-12 linker adopts an "upward" conformation while in the desensitized conformation the linker assumes a "downward" state. It is unclear if a single linker adopting the "downward" state is sufficient to desensitize the entire channel, if all three are needed or some more complex scheme. To accommodate this "downward" state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a tool valuable research tool. Proline substitutions in the chicken ASIC1 ß11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100 to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady state desensitization curves to more acidic pHs while activation curves and ion selectivity were largely unaffected (except for a left shifted activation pH50 of L414P). To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P substitutions only slightly attenuated desensitization, suggesting that conformational changes in the single remaining faster wild type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where ASIC desensitization requires only a single subunit.