A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels.

The Na+-activated K+ channel KNa1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na+ ions bind and increase channel activity is not well understood. Analysis of KNa1.1 channel structures indicate that there is a large twisting of the ßN-αQ loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to potentially form a salt bridge with an aspartate (D839) in ßL in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within a pocket formed by the ßO strand. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found that alanine substitution of selected residues in these regions resulted in almost negligible currents in the presence of up to 40 mM intracellular Na+. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na+. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active KNa1.1, and D884I had Na+ dependence similar to wild-type KNa1.1, while increasing the side-chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na+. We conclude that Na+ binding results in a conformational change that accommodates D884 in the ßO pocket, which triggers further conformational changes in the RCK domains and channel activation.

Is intestinal transport dysfunctional in COVID-19-related diarrhea?

Diarrhea, often severe, is a recognized and frequently early symptom during acute COVID-19 infection and may persist or develop for the first time in patients with long-COVID, with socioeconomic consequences. Diarrheal mechanisms in these cases are poorly understood. There is evidence for disruption of intestinal epithelial barrier function and also for changes in the gut microbiome, which is critical for gut immunity and metabolism. Whether the SARS-CoV-2 virus has adverse effects on intestinal transport proteins is unclear. However, the ability of the virus to inhibit expression and activity of an aldosterone-regulated epithelial sodium (Na+) channel (ENaC) present in human distal colon, which is responsible for Na+ and water salvage, points to possible disruption of other intestinal transport proteins during COVID-19 infection. In this Perspective, we develop this idea by highlighting possible intestinal transport protein targets for the SARS-CoV-2 virus and discussing how their interactions might be explored in the laboratory.

Small molecules restore the function of mutant CLC5 associated with Dent disease.

Dent disease type 1 is caused by mutations in the CLCN5 gene that encodes CLC5, a 2Cl- /H+ exchanger. The CLC5 mutants that have been functionally analysed constitute three major classes based on protein expression, cellular localization and channel function. We tested two small molecules, 4-phenylbutyrate (4PBA) and its analogue 2-naphthoxyacetic acid (2-NOAA), for their effect on mutant CLC5 function and expression by whole-cell patch-clamp and Western blot, respectively. The expression and function of non-Class I CLC5 mutants that have reduced function could be restored by either treatment. Cell viability was reduced in cells treated with 2-NOAA. 4PBA is a FDA-approved drug for the treatment of urea cycle disorders and offers a potential therapy for Dent disease.

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection.

INTRODUCTION: Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%-40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. METHODS: It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl-) channel modulators to investigate their role during the HRSV life-cycle. RESULTS: We demonstrate the requirement for TMEM16A, a calcium-activated Cl- channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. DISCUSSION: These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.

The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility.

Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl- channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily because of the expression of two early regulatory proteins termed small and large tumor antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyV-mediated cancer formation but, thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl- channel modulation can reduce MCPyV ST-induced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST up-regulates two Cl- channels, CLIC1 and CLIC4, which when silenced, inhibit MCPyV ST-induced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are up-regulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease. This highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC.

A cytoplasmic Slo3 isoform is expressed in somatic tissues.

Slo3 is a pH-sensitive and weakly voltage-sensitive potassium channel that is essential for male fertility in mouse and whose expression is regarded as sperm-specific. These properties have proposed Slo3 as a candidate target for male contraceptive drugs. Nonetheless, the tissue distribution of Slo3 expression has not been rigorously studied yet. Applying computational and RT-PCR approaches, we identified expression of two short Slo3 isoforms in somatic mouse tissues such as brain, kidney and eye. These isoforms, which seem to result of transcription starting sites between exons 20 and 21, have an identical open reading frame, both encoding the terminal 381 amino acids of the cytosolic Slo3 domain. We corroborated the expression of these isoforms in mouse brain and testis by Western-blot. The complete isoform encoding the Slo3 ion channel was uniquely detected in testis, both at transcript and protein level. Although the functional role of the cytosolic Slo3 isoforms remains to be established, we propose that they may have a functional effect by modulating Slo channels trafficking and/or activity. This study confirms that expression of full-length Slo3 is sperm-specific but warns against developing contraceptive drugs targeting the C-terminal tail of Slo3 channels.

The changing landscape of membrane protein structural biology through developments in electron microscopy.

Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition, we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins.

Requirement for chloride channel function during the hepatitis C virus life cycle.

Hepatocytes express an array of plasma membrane and intracellular ion channels, yet their role during the hepatitis C virus (HCV) life cycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl(-)) influx that can be inhibited by selective Cl(-) channel blockers. Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl(-) channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl(-) channels during the HCV life cycle.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin.

RATIONALE: Calcium entry is pivotal in the heart and blood vessels, but its significance and mechanisms in adipose tissue are largely unknown. An important factor produced by adipocytes is adiponectin, which confers myocardial protection, insulin-sensitization, and antiatherosclerotic effects. OBJECTIVE: To investigate the relevance of calcium channels to adipocytes and the production of adiponectin. METHODS AND RESULTS: Microarray analysis led to identification of transient receptor potential canonical (TRPC)1 and TRPC5 as channel subunits that are induced when adipocytes mature. Both subunits were found in perivascular fat of patients with atherosclerosis. Intracellular calcium and patch-clamp measurements showed that adipocytes exhibit constitutively active calcium-permeable nonselective cationic channels that depend on TRPC1 and TRPC5. The activity could be enhanced by lanthanum or rosiglitazone, known stimulators of TRPC5 and TRPC5-containing channels. Screening identified lipid modulators of the channels that are relevant to adipose biology. Dietary ω-3 fatty acids (eg, α-linolenic acid) were inhibitory at concentrations that are achieved by ingestion. The adipocyte TRPC1/TRPC5-containing channel was functionally negative for the generation of adiponectin because channel blockade by antibodies, knock-down of TRPC1-TRPC5 in vitro, or conditional disruption of calcium permeability in TRPC5-incorporating channels in vivo increased the generation of adiponectin. The previously recognized capability of α-linolenic acid to stimulate the generation of adiponectin was lost when calcium permeability in the channels was disrupted. CONCLUSIONS: The data suggest that TRPC1 and TRPC5 contribute a constitutively active heteromultimeric channel of adipocytes that negatively regulates adiponectin and through which ω-3 fatty acids enhance the anti-inflammatory adipokine, adiponectin.

Drosophila expressing mutant human KCNT1 transgenes make an effective tool for targeted drug screening in a whole animal model of KCNT1-epilepsy.

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy.

Inhibition of the voltage-gated potassium channel Kv1.5 by hydrogen sulfide attenuates remodeling through S-nitrosylation-mediated signaling.

The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.

Constitutive endocytic recycling and protein kinase C-mediated lysosomal degradation control K(ATP) channel surface density.

Pancreatic ATP-sensitive potassium (K(ATP)) channels control insulin secretion by coupling the excitability of the pancreatic beta-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K(ATP) channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K(ATP) channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K(ATP) channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic beta-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K(ATP) channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic beta-cells, we propose that PKC-regulated K(ATP) channel trafficking may play a role in the regulation of insulin secretion.

Voltage-dependent charge movement associated with activation of the CLC-5 2Cl-/1H+ exchanger.

The family of CLC proteins comprises both Cl(-) channels and Cl(-)/H(+) exchange transporters with varying degrees of voltage dependence. The human CLC-5 is an electrogenic voltage-dependent 2Cl(-)/1H(+) exchanger that gives rise to strongly outwardly rectifying currents when expressed. We conducted whole-cell recordings from HEK293 cells transiently transfected with either wild-type CLC-5 or a permeation-deficient mutant, E268A. With E268A CLC-5 we recorded transient voltage-dependent currents that represent the gating currents associated with CLC-5 activation and had kinetics that could be described by voltage-dependent forward and reverse transition rates. In extracellular solutions rich in Cl(-) or Br(-), CLC-5 exhibited a gating charge of 1.3, but this was reduced to 0.9 in solutions comprising the impermeant anions aspartate, methanesulfonate, sulfate, or HEPES. Extracellular ion depletion by local perfusion with isotonic mannitol failed to reduce the gating charge further. Lowering intracellular pH from 7.4 to 5.4 did not shift the voltage-dependence of the gating currents, but reducing and increasing intracellular Cl(-) shifted the charge-voltage relationship to more negative and positive potentials, respectively. Our data suggest that voltage sensing is an intrinsic property of the CLC-5 protein and that permeant anions, particularly Cl(-), modulate a voltage-dependent transition to an activated state from which Cl(-)/H(+) exchange can occur.

Targeting KNa1.1 channels in KCNT1-associated epilepsy.

Gain-of-function (GOF) pathogenic variants of KCNT1, the gene encoding the largest known potassium channel subunit, KNa1.1, are associated with developmental and epileptic encephalopathies accompanied by severe psychomotor and intellectual disabilities. Blocking hyperexcitable KNa1.1 channels with quinidine, a class I antiarrhythmic drug, has shown variable success in patients in part because of dose-limiting off-target effects, poor blood-brain barrier (BBB) penetration, and low potency. In recent years, high-resolution cryogenic electron microscopy (cryo-EM) structures of the chicken KNa1.1 channel in different activation states have been determined, and animal models of the diseases have been generated. Alongside increasing information about the functional effects of GOF pathogenic variants on KNa1.1 channel behaviour and how they lead to hyperexcitability, these tools will facilitate the development of more effective treatment strategies. We review the range of KCNT1 variants and their functional effects, the challenges posed by current treatment strategies, and recent advances in finding more potent and selective therapeutic interventions for KCNT1-related epilepsies.

Kv1.3 voltage-gated potassium channels link cellular respiration to proliferation through a non-conducting mechanism.

Cellular energy metabolism is fundamental for all biological functions. Cellular proliferation requires extensive metabolic reprogramming and has a high energy demand. The Kv1.3 voltage-gated potassium channel drives cellular proliferation. Kv1.3 channels localise to mitochondria. Using high-resolution respirometry, we show Kv1.3 channels increase oxidative phosphorylation, independently of redox balance, mitochondrial membrane potential or calcium signalling. Kv1.3-induced respiration increased reactive oxygen species production. Reducing reactive oxygen concentrations inhibited Kv1.3-induced proliferation. Selective Kv1.3 mutation identified that channel-induced respiration required an intact voltage sensor and C-terminal ERK1/2 phosphorylation site, but is channel pore independent. We show Kv1.3 channels regulate respiration through a non-conducting mechanism to generate reactive oxygen species which drive proliferation. This study identifies a Kv1.3-mediated mechanism underlying the metabolic regulation of proliferation, which may provide a therapeutic target for diseases characterised by dysfunctional proliferation and cell growth.

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis.

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.

Direct endosomal acidification by the outwardly rectifying CLC-5 Cl(-)/H(+) exchanger.

The voltage-gated Cl(-) channel (CLC) family comprises cell surface Cl(-) channels and intracellular Cl(-)/H(+) exchangers. CLCs in organelle membranes are thought to assist acidification by providing a passive chloride conductance that electrically counterbalances H(+) accumulation. Following recent descriptions of Cl(-)/H(+) exchange activity in endosomal CLCs we have re-evaluated their role. We expressed human CLC-5 in HEK293 cells, recorded currents under a range of Cl(-) and H(+) gradients by whole-cell patch clamp, and examined the contribution of CLC-5 to endosomal acidification using a targeted pH-sensitive fluorescent protein. We found that CLC-5 only conducted outward currents, corresponding to Cl(-) flux into the cytoplasm and H(+) from the cytoplasm. Inward currents were never observed, despite the range of intracellular and extracellular Cl(-) concentrations and pH used. Endosomal acidification in HEK293 cells was prevented by 25 microm bafilomycin-A1, an inhibitor of vacuolar-type H(+)-ATPase (v-ATPase), which actively pumps H(+) into the endosomal lumen. Overexpression of CLC-5 in HEK293 cells conferred an additional bafilomycin-insensitive component to endosomal acidification. This effect was abolished by making mutations in CLC-5 that remove H(+) transport, which result in either no current (E268A) or bidirectional Cl(-) flux (E211A). Endosomal acidification in a proximal tubule cell line was partially sensitive to inhibition of v-ATPase by bafilomycin-A1. Furthermore, in the presence of bafilomycin-A1, acidification was significantly reduced and nearly fully ablated by partial and near-complete knockdown of endogenous CLC-5 by siRNA. These data suggest that CLC-5 is directly involved in endosomal acidification by exchanging endosomal Cl(-) for H(+).

CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Structure-Based Identification and Characterization of Inhibitors of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel.

Drug-resistant epileptic encephalopathies of infancy have been associated with KCNT1 gain-of-function mutations, which increase the activity of KNa1.1 sodium-activated potassium channels. Pharmacological inhibition of hyperactive KNa1.1 channels by quinidine has been proposed as a stratified treatment, but mostly this has not been successful, being linked to the low potency and lack of specificity of the drug. Here we describe the use of a previously determined cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify how quinidine binds to the channel pore and, using computational methods, screened for compounds predicated to bind to this site. We describe six compounds that inhibited KNa1.1 channels with low- and sub-micromolar potencies, likely also through binding in the intracellular pore vestibule. In hERG inhibition and cytotoxicity assays, two compounds were ineffective. These may provide starting points for the development of new pharmacophores and could become tool compounds to study this channel further.