Modulation Of Channel Function Research Articles

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region

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 (NaVs and CaVs, respectively). Unlike NaVs and CaVs, 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 NaV, CaV, 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.

Aquaporin splice variation differentially modulates channel function during marine teleost egg hydration.

Aquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.

Site and Mechanism of ML252 Inhibition of Kv7 Voltage-Gated Potassium Channels.

Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.

Voltage sensor targeted activation of Kv7 channels by QO-58.

Kv7 (KCNQ) voltage-gated potassium channels are key modulators of neuronal excitability and have been a promising target for developing novel drugs to treat epilepsy and pain. Drug discovery has focused on identifying small molecules that modulate channel function and understanding their mechanisms of action. Special attention has been given to Kv7 channel activators, which may have therapeutic benefits in epilepsy or pain. In this study, we compared the effects and mechanism of a candidate Kv7 activator, QO-58, which has been previously described but has an unclear site and mechanism of action. We compared QO-58 actions with other well-characterized activators that influence either the VSD (ICA-069673) or pore domain (ML213) of Kv7 channels. QO-58 exhibits strong subtype specificity for Kv7.2 over Kv7.3, similar to VSD-targeted activators. Additionally, QO-58 exhibits prominent state-dependent actions, as it requires channels to activate before binding, and dramatically decelerates closure of Kv7.2. We also tested the effects of multiple VSD mutations on QO-58 and ICA-069673 actions. Interestingly, we found that the Kv7.2[F168L] mutation, previously shown to abolish ICA-069673 sensitivity, does not markedly affect QO-58 mediated effects. Notably, Kv7.2[I134A] and Kv7.2[I173T] weaken the effects of ICA-069673, but are hypersensitive to QO-58. This finding may suggest that dimensions of the VSD binding pocket influence the potency of smaller compounds (e.g. ICA-069673) relative to bulkier ones (e.g. QO-58), despite overlap in their binding sites. Our findings highlight that key contacts underlying sensitivity to VSD-targeted activators may vary significantly depending on the chemical and steric features of the drug. We anticipate that further investigation of VSD-targeted activators will continue to clarify the complex pharmacophore of the VSD binding pocket in Kv7 channels.

Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction.

Most mechanically gated channels are sensitive to force translated through the membrane. The lipid bilayer can modulate channel function directly through lipid/protein interactions or indirectly based on membrane mechanical properties. Cholesterol is an important component of the membrane and a major modulator of membrane mechanical properties. While other ion channels and mechanically gated channels can be activated or modulated by changes to the membrane cholesterol, the functional role of membrane cholesterol in regulation of mammalian cochlear mechanotransduction (MET) channels has not been closely investigated. Using whole-cell patch clamping and live-cell fluorescence lifetime imaging (FLIM) of a viscosity-sensitive molecular rotor BODIPY 1c for the first time in the inner ear, we examined the role of membrane cholesterol in modulating the MET response properties of rat cochlear hair cells. Molecular rotors are fluorophores for which the fluorescence lifetime (the average time a fluorophore remains in the excited state) increase with increasing viscosity of their immediate environment. We confirmed extraction of cholesterol, using methyl β cyclodextrin (MβCD), with reduced filipin staining in both inner and outer hair bundles. MβCD results in reversible reduction in fluorescence lifetime in hair bundles, suggesting initial reduction followed by gradual recovery in the stereocilia membrane viscosity. MβCD reversibly increases the channel resting open probability, suggesting that cholesterol depletion increases force transfer to the MET channel. Together this data suggests that the cell membrane is part of the force relay machinery to the MET channel and could possibly interact directly with components of the MET machinery. Further studies are needed to generate causal link between MET channel gating and membrane mechanics.

Molecular simulation of the Kv7.4[ΔS269] mutant channel reveals that ion conduction in the cavity is perturbed due to hydrophobic gating

Mutations in the voltage-gated potassium channel Kv7.4 (encoded as KCNQ4) lead to the early onset of non-syndromic hearing loss, which is significant during language acquisition. The deletion of the S269 pore residue (genetic Δ mutation) in Kv7.4 has been reported to be associated with hearing loss. So far, there is no mechanistic understanding of how this mutation modulates channel function. To understand the role of S269 in ion conduction, we performed molecular dynamics simulations for both wild type and ΔS269 mutant channels. Simulations indicate that the ΔS269 mutation suppresses the fluctuations in the neighboring Y269 residue and thereby consolidates the ring formed by I307 and F310 residues in the adjacent S6 helixes in the cavity region. We show that the long side chains of I307 near the entrance to the cavity form a hydrophobic gate. Comparison of the free energy profiles of a cavity ion in Kv7.4 and Kv7.4[ΔS269] channels reveals a sizable energy barrier in the latter case, which suppresses ion conduction. Thus the simulation studies reveal that the hydrophobic gate resulting from the ΔS269 mutation appears to be responsible for sensorineural hearing loss.

Role of High-Throughput Electrophysiology in Drug Discovery.

Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high-throughput electrophysiology and its applications in drug discovery is provided. © 2019 The Authors.

Amphipathic molecules modulate PIEZO1 activity.

PIEZO proteins are large eukaryotic mechanically-gated channels that function as homotrimers. The basic PIEZO1 structure has been elucidated by CryoEM and it assembles into a protein-lipid dome. A curved lipid region allows for the transition to the lipid bilayer from the dome (footprint). Gating PIEZO1 is mediated by bilayer tension that induces an area change in the lipid dome. The footprint region is thought to be energetically important for changes in lateral tension. Amphipathic molecules can modulate channel function beyond the intrinsic gating properties of PIEZO1. As a result, molecules that modify lipid properties within the lipid-channel complex (footprint and dome) will profoundly affect channel kinetics. In this review, we summarize the effects some amphipathic molecules have on the lipid bilayer and PIEZO1 function. PIEZO1 has three states, closed, open and inactivated and amphipathic molecules influence these transitions. The amphipathic peptide, GsMTx4, inhibits the closed to open transition. While saturated fatty acids also prevent PIEZO1 gating, the effect is mediated by stiffening the lipids, presumably in both the dome and footprint region. Polyunsaturated fatty acids can increase disorder within the lipid-protein complex affecting channel kinetics. PIEZO1 can also form higher-ordered structures that confers new kinetic properties associated with clustered channels. Cholesterol-rich domains house PIEZO1 channels, and depletion of cholesterol causes a breakdown of those domains with changes to channel kinetics and channel diffusion. These examples underscore the complex effects lipophilic molecules can have on the PIEZO1 lipid dome structure and thus on the mechanical response of the cell.

Phosphorylation of the HCN channel auxiliary subunit TRIP8b is altered in an animal model of temporal lobe epilepsy and modulates channel function

Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser237 of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser237 is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b-HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.

Molecular determinants of chemical modulation of two-pore domain potassium channels.

The K+ ion channels comprising the two-pore domain (K2P) family have specific biophysical roles in generating the critical regulatory K+ current. Ion flow through K2P channels and, implicitly, channel regulation is mediated by diverse metabolic and physical inputs such as mechanical stimulation, interaction with lipids or endogenous regulators, intra- or extracellular pH, and phosphorylation, while their function can be finely tuned by chemical compounds. In the latter category, some drug-channel interactions can lead to sideeffects or have clinical action, while identifying novel chemical modulators of K2Ps is an area of intense research. Due to their cellular and therapeutic importance, much attention was turned to these channels in recent years and several experimental approaches have pinpointed the molecular determinants of K2P chemical modulation. Given their unique structural features and properties, chemical modulators act on K2P channels in multiple and diverse ways. In this review, the particularities of K2P modulation by chemical compounds, such as binding modality, affinity, or position, are identified, synthesized, and linked to structural and functional properties in order to refer to how activators and blockers modify channel function and vice versa, focusing on specificity related to protein structure (and its modification) and cross-linking information among different subfamilies.

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid.

Calcium modulation of cardiac sodium channels.

Modification of voltage-gated Na+ channel (NaV ) function by intracellular Ca2+ has been a topic of much controversy. Early studies relied on measuring NaV function in the absence or presence of intracellular Ca2+ , and generated seemingly disparate results. Subsequent investigations revealed the mechanism(s) of Ca2+ -driven NaV modulation are complex and involve multiple accessory proteins. The Ca2+ -sensing protein calmodulin (CaM) has a central role in tuning NaV function to [Ca2+ ]i , but the mechanism has been obscured by other proteins (such as fibroblast growth factors (FGF) or CaM-dependent kinase II (CaMKII)) that can also modify channel function or exert an influence in a Ca2+ -dependent manner. Significant progress has been made in understanding the architecture of full-length ion channels and the structural and biophysical details of NaV -accessory protein interactions. Interdisciplinary structure-function studies are beginning to resolve the effect each interaction has on NaV gating. Carefully designed structure-guided or strategically selected disease-associated mutations are able to impair NaV -accessory protein interactions without altering other properties of channel function. Recently CaM was found to engage part of NaV 1.5 that is required for channel inactivation with high affinity. Careful impairment of this interaction disrupted NaV 1.5's ability to recover from inactivation. Such results support a paradigm of CaM-facilitated recovery from inactivation (CFRI). How NaV -CaM, CaMKII and FGF/fibroblast growth factor homologous factor interactions affect the timing or function of CFRI in cardiomyocytes remain open questions that are discussed herein. Moreover whether CFRI dysfunction or premature activation underlie certain NaV channelopathies are important questions that will require further investigation.

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273–317) and end-tail (aa 318–333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.

Identification of IQM-266, a Novel DREAM Ligand That Modulates KV4 Currents.

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (IA). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (IA) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge () at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of IA. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal IA in pathological processes.

Pharmacological modulation of mitochondrial ion channels

The field of mitochondrial ion channels has undergone a rapid development during the last three decades, due to the molecular identification of some of the channels residing in the outer and inner membranes. Relevant information about the function of these channels in physiological and pathological settings was gained thanks to genetic models for a few, mitochondria-specific channels. However, many ion channels have multiple localizations within the cell, hampering a clear-cut determination of their function by pharmacological means. The present review summarizes our current knowledge about the ins and outs of mitochondrial ion channels, with special focus on the channels that have received much attention in recent years, namely, the voltage-dependent anion channels, the permeability transition pore (also called mitochondrial megachannel), the mitochondrial calcium uniporter and some of the inner membrane-located potassium channels. In addition, possible strategies to overcome the difficulties of specifically targeting mitochondrial channels versus their counterparts active in other membranes are discussed, as well as the possibilities of modulating channel function by small peptides that compete for binding with protein interacting partners. Altogether, these promising tools along with large-scale chemical screenings set up to identify new, specific channel modulators will hopefully allow us to pinpoint the actual function of most mitochondrial ion channels in the near future and to pharmacologically affect important pathologies in which they are involved, such as neurodegeneration, ischaemic damage and cancer. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

Characterization of Functional Effects of Two New Active Fractions Isolated From Scorpion Venom on Neuronal Ca2+ Spikes: A Possible Action on Ca2+-Dependent Dependent K+ Channels

Introduction:It is a long time that natural toxin research is conducted to unlock the medical potential of toxins. Although venoms-toxins cause pathophysiological conditions, they may be effective to treat several diseases. Since toxins including scorpion toxins target voltage-gated ion channels, they may have profound effects on excitable cells. Therefore, elucidating the cellular and electrophysiological impacts of toxins, particularly scorpion toxins would be helpful in future drug development opportunities.Methods:Intracellular recording was made from F1 cells of Helix aspersa in the presence of calcium Ringer solution in which Na+ and K+ channels were blocked. Then, the modulation of channel function in the presence of extracellular application of F4 and F6 toxins and kaliotoxin (KTX; 50 nM and 1 μM) was examined by assessing the electrophysiological characteristics of calcium spikes.Results:The two active toxin fractions, similar to KTX, a known Ca2+-activated K+ channel blocker, reduced the amplitude of AHP, enhanced the firing frequency of calcium spikes and broadened the duration of Ca2+ spikes. Therefore, it might be inferred that these two new fractions induce neuronal hyperexcitability possibly, in part, by blocking calcium-activated potassium channel current. However, this supposition requires further investigation using voltage clamping technique.Conclusion:These toxin fractions may act as blocker of calcium-activated potassium channels.

Characterization of Functional Effects of Two New Active Fractions Isolated From Scorpion Venom on Neuronal Ca2+ Spikes: A Possible Action on Ca2+-Dependent Dependent K+ Channels.

Introduction:Based on the literature, unihemispheric concurrent dual-site anodal transcranial Direct Current Stimulation (a-tDCSUHCDS) of primary Motor cortex (M1) and Dorsolateral Prefrontal Cortex (DLPFC) would be more efficient than conventional a-tDCS of M1 to induce larger and longer-lasting M1 corticospinal excitability. The main objective of the present study was to compare the effects of a-tDCSUHCDS and conventional M1 a-tDCS on the extent and durability of the motor sequence acquisition in healthy individuals.Methods:In this randomized sham-controlled study, healthy volunteers were randomly divided into three groups: experimental (a-tDCSUHCDS), control (M1 a-tDCS), and sham stimulation groups. The participants practiced serial response time task over three consecutive days when they simultaneously received a-tDCS. Using the skill measure, we assessed motor learning up to 4 weeks after the completion of experimental conditions.Results:Data analysis revealed that all groups exhibited the improved trend over the training course (P<0.001). There were no significant differences in skill acquisition among groups at post-intervention (P>0.05), while a significant improvement was observed between experimental and sham group at the retention time (P<0.05). Moreover, there were no significant differences between the control and two other groups with regard to the retention time (P>0.05).Conclusion:These results revealed a significant increase in the skill acquisition by a-tDCSUHCDS technique with regard to retention issue, which could be a valuable finding in neuro-rehabilitation field.

Epithelial sodium channel biogenesis and quality control in the early secretory pathway.

The epithelial sodium channel, ENaC, is responsible for Na reabsorption in several epithelia and is composed of homologous α, β, and γ subunits. Here, we will explore the differential regulation of ENaC subunits during biogenesis in the early secretory pathway. ENaC subunits are subject to numerous posttranslational modifications, including glycosylation, protease activation, disulfide bond formation, palmitoylation, and glycosylation, each of which modulate channel function. For example, glycan addition is regulated by sodium and affects protease activation at the cell surface, protein trafficking, sodium-dependent regulation, and sodium transport. Glycosylation of the α subunit also determines whether a chaperone, Lhs1/GRP170, selects the protein for endoplasmic reticulum-associated degradation. Recognition by this chaperone is blocked by assembly of the ENaC transmembrane domains. In contrast, cytosolic lysines are acetylated in the early secretory pathway, which inhibits ubiquitination and endocytosis at the cell surface. As sodium reabsorption by ENaC in the distal nephron regulates salt and water homeostasis, ENaC function is critical for human health. Therefore, identifying and characterizing modifiers of ENaC in the early secretory pathway may provide both new therapeutic targets and further our basic understanding of membrane protein assembly and regulation.

A Mechanism of Calmodulin Modulation of the Human Cardiac Sodium Channel

The function of the human cardiac sodium channel (NaV1.5) is modulated by the Ca2+ sensor calmodulin (CaM), but the underlying mechanism(s) are controversial and poorly defined. CaM has been reported to bind in a Ca2+-dependent manner to two sites in the intracellular loop that is critical for inactivation of NaV1.5 (inactivation gate [IG]). The affinity of CaM for the complete IG was significantly stronger than that of fragments that lacked both complete binding sites. Structural analysis by nuclear magnetic resonance, crystallographic, and scattering approaches revealed that CaM simultaneously engages both IG sites using an extended configuration. Patch-clamp recordings for wild-type and mutant channels with an impaired CaM-IG interaction revealed CaM binding to the IG promotes recovery from inactivation while impeding the kinetics of inactivation. Models of full-length NaV1.5 suggest that CaM binding to the IG directly modulates channel function by destabilizing the inactivated state, which would promote resetting of the IG after channels close.

Mechanosensitive Ion Channels: TRPV4 and P2X7 in Disseminating Cancer Cells.

Cancer metastasis is the second leading cause of death in the United States. Despite its morbidity, metastasis is an inefficient process that few cells can survive. However, cancer cells can overcome these metastatic barriers via cellular responses to microenvironmental cues, such as through mechanotransduction. This review focuses on the mechanosensitive ion channels TRPV4 and P2X7, and their roles in metastasis, as both channels have been shown to significantly affect tumor cell dissemination. Upon activation, these channels help form tumor neovasculature, promote transendothelial migration, and increase cell motility. Conversely, they have also been linked to forms of cancer cell death dependent upon levels of activation, implying the complex functionality of mechanosensitive ion channels. Understanding the roles of TRPV4, P2X7 and other mechanosensitive ion channels in these processes may reveal new possible drug targets that modify channel function to reduce a tumor's metastatic potential.