Wild-type Channels Research Articles

Multiple ALMT subunits combine to form functional anion channels: A case study for rice ALMT7

The Aluminum Activated Malate Transporter (ALMT) family members are anion channels that play important roles in organic acid transport, stress resistance, growth, development, fertilization and GABA responses. The rice malate permeable OsALMT7 influences panicle development and grain yield. A truncated OsALMT7 mutant, panicle apical abortion1 (paab1) lacking at least 2 transmembrane helices, mediates reduced malate efflux resulting in yield reducing. Here, we further investigated the contribution of OsALMT7 transmembrane helices to channel activity, using heterologous expression in Xenopus laevis oocytes. We further found that OsALMT7 formed as a homomer by co-expressing OsALMT7 and paab1 proteins in oocytes and detecting the physical interaction between two OsALMT7, and between OsALMT7 and paab1 mutant protein. Further study proved that not just OsALMT7, mutants of TaALMT1 inhibit wild-type TaALMT1 channel, indicating that ALMTs might perform channel function as homomers. Our discovery brings a light for ion channel structure and homomultimer regulation understanding for ALMT anion channels and potential for crop grain yield and stress response improvement in the context of the essential role of ALMTs in these plant processes.

TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors

Communication between TRPC channels and IP3 receptors (IP3R) is considered pivotal in the generation of spatiotemporal Ca2+signaling patterns. Here we revisited the role of TRPC3-IP3R coupling for local Ca2+ signaling within TRPC3-harbouring micro/nanodomains using R-GECO as a reporter, fused to the channel´s C-terminus. Cytoplasmic Ca2+ changes at TRPC3 originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes at TRPC3 channels expressed in HEK293 cells were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by point mutations, which impair Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted microdomain Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. Similarly, when extracellular Ca2+ was omitted, IP3R-mediated Ca2+ transients and Ca2+ oscillations were promoted at the cytoplasmic face of wild-type TRPC3 channels. By contrast, oscillations, as well as initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by preexposure to low-level PLC activity. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. We suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to determine Ca2+ signaling signatures and enable specificity of downstream signaling.

De novo Y1460C missense variant in NaV1.1 impedes the pore region and results in epileptic encephalopathy

Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures. SCN1A encodes NaV1.1, a neuronal voltage-gated Na+ channel that is highly expressed throughout the central nervous system. NaV1.1 is localized within the axon initial segment where it plays a critical role in the initiation and propagation of action potentials and neuronal firing, predominantly in γ-amino-butyric-acid (GABA)ergic neurons of the hippocampus. The objective of this study was to characterize a de novo missense variant of uncertain significance in the SCN1A gene of a proband presented with febrile status epilepticus characterized by generalized tonic clonic movements associated with ictal emesis and an abnormal breathing pattern. Screening a gene panel revealed a heterozygous missense variant of uncertain significance in the SCN1A gene, designated c.4379A>G, p.(Tyr1460Cys). The NaV1.1 wild-type (WT) and mutant channel reproduced in vivo and were transfected in HEK 293 cells. Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. This NaV1.1 variant (Tyr1460Cys) failed to express functional Na+ currents when expressed in HEK293 cells, most probably due to a pore defect of the channel given that the cell surface expression of the channel was normal. Currents generated after co-transfection with functional WT channels exhibited biophysical properties comparable to those of WT channels, which was mainly due to the functional WT channels at the cell surface. The NaV1.1 variant failed to express functional Na+ currents, most probably due to pore impairment and exhibited a well-established loss of function mechanism. The present study highlights the added-value of functional testing for understanding the pathophysiology and potential treatment decisions for patients with undiagnosed developmental epileptic encephalopathy.

Germline de novo variant F747S extends the phenotypic spectrum of CACNA1D Ca2+ channelopathies.

Germline gain-of-function missense variants in the pore-forming Cav1.3 α1-subunit (CACNA1D gene) confer high risk for a severe neurodevelopmental disorder with or without endocrine symptoms. Here, we report a 4-week-old new-born with the novel de novo missense variant F747S with a so far not described prominent jittering phenotype in addition to symptoms previously reported for CACNA1D mutations including developmental delay, elevated aldosterone level and transient hypoglycemia. We confirmed the pathogenicity of this variant in whole-cell patch-clamp experiments with wild-type and F747S mutant channels heterologously expressed together with α2δ1 and cytosolic β3 or membrane-bound β2a subunits. Mutation F747S caused the quantitatively largest shift in the voltage dependence of activation (-28mV) reported so far for CACNA1D germline mutations. It also shifted inactivation to more negative voltages, slowed the time course of current inactivation and slowed current deactivation upon repolarization with both co-expressed β-subunits. In silico modelling and molecular docking, simulations revealed that this gain-of-function phenotype can be explained by formation of a novel inter-domain hydrogen bond between mutant residues S747 (IIS6) with N1145 (IIIS6) stabilizing selectively the activated open channel state. F747S displayed 2-6-fold increased sensitivity for the L-type Ca2+ channel blocker isradipine compared to wild type. Our data confirm the pathogenicity of the F747S variant with very strong gain-of-function gating changes, which may contribute to the novel jittering phenotype. Increased sensitivity for isradipine suggests this drug for potential symptomatic off-label treatment for carriers of this mutation.

Integrin-KCNB1 potassium channel complexes regulate neocortical neuronal development and are implicated in epilepsy

Potassium (K+) channels are robustly expressed during prenatal brain development, including in progenitor cells and migrating neurons, but their function is poorly understood. Here, we investigate the role of voltage-gated K+ channel KCNB1 (Kv2.1) in neocortical development. Neuronal migration of glutamatergic neurons was impaired in the neocortices of KCNB1 null mice. Migratory defects persisted into the adult brains, along with disrupted morphology and synaptic connectivity. Mice developed seizure phenotype, anxiety, and compulsive behavior. To determine whether defective KCNB1 can give rise to developmental channelopathy, we constructed Knock In (KI) mice, harboring the gene variant Kcnb1R312H (R312H mice) found in children with developmental and epileptic encephalopathies (DEEs). The R312H mice exhibited a similar phenotype to the null mice. Wild type (WT) and R312H KCNB1 channels made complexes with integrins α5β5 (Integrin_K+ channel_Complexes, IKCs), whose biochemical signaling was impaired in R312H brains. Treatment with Angiotensin II in vitro, an agonist of Focal Adhesion kinase, a key component of IKC signaling machinery, corrected the neuronal abnormalities. Thus, a genetic mutation in a K+ channel induces severe neuromorphological abnormalities through non-conducting mechanisms, that can be rescued by pharmacological intervention. This underscores a previously unknown role of IKCs as key players in neuronal development, and implicate developmental channelopathies in the etiology of DEEs.

Utility of in vivo zebrafish cardiac assay to predict the functional impact of KCNQ1 variants

Abstract Background Genetic testing for inherited arrhythmias and discriminating pathogenic from benign variants are integral for the gene-based medicine. However, the high throughput in vivo functional analysis for the rare variants of the KCNQ1 potassium channel is scarce. Purpose We tested the utility of the in vivo zebrafish cardiac assay for determining the pathogenicity of the KCNQ1 variants identified in patients with long QT syndrome (LQTS) and atrial fibrillation (AF). Methods We generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the KCNQ1 homolog in zebrafish (kcnq1+/+). To test the utility of the cardiac assay, we used five KCNQ1 variants identified in patients with LQTS or familial AF. Human wild-type or mutant KCNQ1 cRNA (Q1) was co-injected with human KCNE1 cRNA (E1) into the F3 generation embryos with homozygous deletions. We dissected the hearts from the thorax at 72 hour-post-fertilization and measured transmembrane potential in zebrafish heart using the disrupted patch technique. Action potential duration was calculated as the time interval between the peak maximum upstroke velocity and 90% of repolarization (APD90). We compared the APD90s with patients' clinical phenotype and IKs density measured by patch-clamp technique in heterologous system. Results The mean APD90 of embryos with kcnq1del/del was 279±48 ms, which was restored by injecting Q1 WT and E1 (159±29 ms) to that with kcnq1+/+ (167±28 ms). We tested if the mean APD90 of embryos with kcnq1del/del was restored (shortened) by injecting the KCNQ1 variants. First we tested the dominant negative variant p.S277L and the trafficking deficient variant p.T587M. Patients with these variants showed significant prolonged QT intervals, and patch clamp study showed both variants caused the non-functional channels. Zebrafish cardiac assay showed the mean APD90 of embryos with kcnq1del/del+ Q1 S277L+E1 or Q1 T587M+E1 was significantly longer than that with kcnq1del/del+Q1 WT+E1 (Table). Next we tested in-frame variant c.1472_1473 ins GGACCT, which was identified from a patient with AF and normal QT interval. Patch clamp study showed the current density of the mutant KCNQ1 channel with KCNE1 was comparable to that of wild-type KCNQ1 channel with KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del shortened by injecting Q1 insACCTGG +E1 (Table). Finally we tested a missense variant p.R451Q, which was identified from a patient with LQTS. Patch clamp study showed the currents in the cells transfected with R451Q+KCNE1 were similar to those with WT+KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del+Q1 R451Q+E1 was longer than that with kcnq1del/del+Q1 WT+E1 (Table). Conclusions Functional analysis of in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with LQTS. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Grant-in-Aid for Scientific Research (C)

Three dimensional modelling of mutant Kir2.1 channel PIP2 interactions help stratify arrhythmia severity in Andersen Tawil syndrome type 1

Abstract Background Andersen-Tawil type 1 (ATS1) is associated with loss-of-function mutations in the inward rectifier potassium channel Kir2.1, which controls cardiac excitability and impulse conduction. Phosphatidylinositol-4,5-bisphosphate (PIP2) acts as an essential cofactor regulating the opening of Kir2.1 channels. Fifty percent of reported ATS1 mutations affect Kir2.1-PIP2 interactions, leading to ECG defects, ventricular arrhythmias and sudden cardiac death (SCD) by mechanisms that are poorly understood. Purpose To test the hypothesis that the degree of arrhythmogenic severity of ATS1 mutations disrupting PIP2-Kir2.1 binding may be predicted by the level of polarization of the mutant Kir2.1 channel pore. Methods We first used a statistical mean value approach to classify the 40 known arrhythmogenic ATS1 mutations impacting Kir2.1-PIP2 interaction (N=260 individuals) according to arrhythmogenic severity, ranging from SCD through ventricular bigeminy and QT prolongation. We then generated 3D in-silico atomic models of the wildtype channel and the 10 mutant channels with the most severe arrhythmic phenotype to assess the mechanism of the structural defects associated with Kir2.1-PIP2 disruption. Results Our cardiac lethality scoring stratifying Kir2.1 mutations according to arrhythmogenic severity was validated by three additional biostatical quantitative measures. On in-silico modelling, wildtype Kir2.1 channels without PIP2 binding had transmembrane and cytoplasmic pore radius of 1.5 and 3 Å, respectively. Kir2.1-PIP2 interactions increased transmembrane and cytoplasmic pore radius to 3 and 6 Å, respectively. All 10 Kir2.1 mutations had similar transmembrane and cytoplasmic pore radius of ∼1.0 and ∼3.0 Å, respectively. The most severe mutations yielded pore channels with highly polarized electrostatic forces. Remarkably, simulations showed a descending electrostatic pattern at the transmembrane region of PIP2 binding, where the more severe the mutation, the more positive that region was. Structural changes produced by mutations correlated with cardiac severity (R2=0.51; p<0.005) in that the most drastically altered protein structure correlated with the most severe arrhythmic phenotype. Conclusions Computer simulations of mutant Kir2.1 channel structure from the most arrhythmogenic to the least arrhythmogenic predict a gradual decrease in polarization of electrostatic forces along the Kir2.1 channel pore. The results reveal a novel mechanistic stratification of arrhythmogenic severity of ATS1 mutant Kir2.1 channel-PIP2 interactions and open new pathways for developing more personalized ATS1 patient therapies. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): La Caixa Banking Foundation under the project code HR18-00304Fundaciόn La Marato TV3: Ayudas a la investigaciόn en enfermedades raras 2020

The nonconducting W434F mutant adopts upon membrane depolarization an inactivated-like state that differs from wild-type Shaker-IR potassium channels.

Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

Loss-of-function variants in the KCNQ5 gene are implicated in genetic generalized epilepsies.

SummaryBackgroundDe novo missense variants in KCNQ5, encoding the voltage-gated K+ channel KV7.5, have been described to cause developmental and epileptic encephalopathy (DEE) or intellectual disability (ID). We set out to identify disease-related KCNQ5 variants in genetic generalized epilepsy (GGE) and their underlying mechanisms.Methods1292 families with GGE were studied by next-generation sequencing. Whole-cell patch-clamp recordings, biotinylation and phospholipid overlay assays were performed in mammalian cells combined with homology modelling.FindingsWe identified three deleterious heterozygous missense variants, one truncation and one splice site alteration in five independent families with GGE with predominant absence seizures; two variants were also associated with mild to moderate ID. All missense variants displayed a strongly decreased current density indicating a loss-of-function (LOF). When mutant channels were co-expressed with wild-type (WT) KV7.5 or KV7.5 and KV7.3 channels, three variants also revealed a significant dominant-negative effect on WT channels. Other gating parameters were unchanged. Biotinylation assays indicated a normal surface expression of the variants. The R359C variant altered PI(4,5)P2-interaction.InterpretationOur study identified deleterious KCNQ5 variants in GGE, partially combined with mild to moderate ID. The disease mechanism is a LOF partially with dominant-negative effects through functional deficits. LOF of KV7.5 channels will reduce the M-current, likely resulting in increased excitability of KV7.5-expressing neurons. Further studies on network level are necessary to understand which circuits are affected and how this induces generalized seizures.FundingDFG/FNR Research Unit FOR-2715 (Germany/Luxemburg), BMBF rare disease network Treat-ION (Germany), foundation ‘no epilep’ (Germany).

Clinically Relevant KCNQ1 Variants Causing KCNQ1-KCNE2 Gain-of-Function Affect the Ca2+ Sensitivity of the Channel.

Dominant KCNQ1 variants are well-known for underlying cardiac arrhythmia syndromes. The two heterozygous KCNQ1 missense variants, R116L and P369L, cause an allelic disorder characterized by pituitary hormone deficiency and maternally inherited gingival fibromatosis. Increased K+ conductance upon co-expression of KCNQ1 mutant channels with the beta subunit KCNE2 is suggested to underlie the phenotype; however, the reason for KCNQ1-KCNE2 (Q1E2) channel gain-of-function is unknown. We aimed to discover the genetic defect in a single individual and three family members with gingival overgrowth and identified the KCNQ1 variants P369L and V185M, respectively. Patch-clamp experiments demonstrated increased constitutive K+ conductance of V185M-Q1E2 channels, confirming the pathogenicity of the novel variant. To gain insight into the pathomechanism, we examined all three disease-causing KCNQ1 mutants. Manipulation of the intracellular Ca2+ concentration prior to and during whole-cell recordings identified an impaired Ca2+ sensitivity of the mutant KCNQ1 channels. With low Ca2+, wild-type KCNQ1 currents were efficiently reduced and exhibited a pre-pulse-dependent cross-over of current traces and a high-voltage-activated component. These features were absent in mutant KCNQ1 channels and in wild-type channels co-expressed with calmodulin and exposed to high intracellular Ca2+. Moreover, co-expression of calmodulin with wild-type Q1E2 channels and loading the cells with high Ca2+ drastically increased Q1E2 current amplitudes, suggesting that KCNE2 normally limits the resting Q1E2 conductance by an increased demand for calcified calmodulin to achieve effective channel opening. Our data link impaired Ca2+ sensitivity of the KCNQ1 mutants R116L, V185M and P369L to Q1E2 gain-of-function that is associated with a particular KCNQ1 channelopathy.

Clinical comparison and functional study of the L703P: a recurrent mutation in human SCN4A that causes sodium channel myotonia

The non-dystrophic myotonias are inherited skeletal muscle disorders characterized by skeletal muscle stiffness after voluntary contraction, without muscle atrophy. Based on their clinical features, non-dystrophic myotonias are classified into myotonia congenita, paramyotonia congenita, and sodium channel myotonia. Using whole-exome next-generation sequencing, we identified a L703P mutation (c.2108T>C, p.L703P) in SCN4A in a Chinese family diagnosed with non-dystrophic myotonias. The clinical findings of patients in this family included muscle stiffness and hypertrophy. The biophysical properties of wildtype and mutant channels were investigated using whole-cell patch clamp. L703P causes both gain-of-function and loss-of-function changes in Nav1.4 properties, including decreased current density, impaired recovery, enhanced activation and slow inactivation. Our study demonstrates that L703P is a pathogenic variant for myotonia, and provides additional electrophysiological information for understanding the pathogenic mechanism of SCN4A-associated channelopathies.

Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita.

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in “gating-defective” or “expression-defective” mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.

Investigation of CACNA1I Cav3.3 Dysfunction in Hemiplegic Migraine.

Familial hemiplegic migraine (FHM) is a severe neurogenetic disorder for which three causal genes, CACNA1A, SCN1A, and ATP1A2, have been implicated. However, more than 80% of referred diagnostic cases of hemiplegic migraine (HM) are negative for exonic mutations in these known FHM genes, suggesting the involvement of other genes. Using whole-exome sequencing data from 187 mutation-negative HM cases, we identified rare variants in the CACNA1I gene encoding the T-type calcium channel Cav3.3. Burden testing of CACNA1I variants showed a statistically significant increase in allelic burden in the HM case group compared to gnomAD (OR = 2.30, P = 0.00005) and the UK Biobank (OR = 2.32, P = 0.0004) databases. Dysfunction in T-type calcium channels, including Cav3.3, has been implicated in a range of neurological conditions, suggesting a potential role in HM. Using patch-clamp electrophysiology, we compared the biophysical properties of five Cav3.3 variants (p.R111G, p.M128L, p.D302G, p.R307H, and p.Q1158H) to wild-type (WT) channels expressed in HEK293T cells. We observed numerous functional alterations across the channels with Cav3.3-Q1158H showing the greatest differences compared to WT channels, including reduced current density, right-shifted voltage dependence of activation and inactivation, and slower current kinetics. Interestingly, we also found significant differences in the conductance properties exhibited by the Cav3.3-R307H and -Q1158H variants compared to WT channels under conditions of acidosis and alkalosis. In light of these data, we suggest that rare variants in CACNA1I may contribute to HM etiology.

Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG.

Long QT syndrome type II (LQT2) is caused by loss‐of‐function mutations in the hERG K+ channel, leading to increased incidence of cardiac arrest and sudden death. Many genetic variants have been reported in the hERG gene with various consequences on channel expression, permeation, and gating. Only a small number of LQT2 causing variants has been characterized to define the underlying pathophysiological causes of the disease. We sought to determine the characteristics of the frameshift variant p.Thr1019ProfsX38 (T1019PfsX38) which affects the C‐terminus of the protein. This mutation was identified in an extended Omani family of LQT2. It replaces the last 140 amino acids of hERG with 37 unique amino acids. T1019 is positioned at a distinguished region of the C‐terminal tail of hERG, as predicted from the deep learning system AlphaFold v2.0. We employed the whole‐cell configuration of the patch‐clamp technique to study wild‐type and mutant channels that were transiently expressed in human embryonic kidney 293 (HEK293) cells. Depolarizing voltages elicited slowly deactivating tail currents that appeared upon repolarization of cells that express either wild‐type‐ or T1019PfsX38‐hERG. There were no differences in the voltage and time dependencies of activation between the two variants. However, the rates of hERG channel deactivation at hyperpolarizing potentials were accelerated by T1019PfsX38. In addition, the voltage dependence of inactivation of T1019PfsX38‐hERG was shifted by 20 mV in the negative direction when compared with wild‐type hERG. The rates of channel inactivation were increased in the mutant channel variant. Next, we employed a step‐ramp protocol to mimic membrane repolarization by the cardiac action potential. The amplitudes of outward currents and their integrals were reduced in the mutant variant when compared with the wild‐type variant during repolarization. Thus, changes in the gating dynamics of hERG by the T1019PfsX38 variant contribute to the pathology seen in affected LQT2 patients.

Novel HCN1 Mutations Associated With Epilepsy and Impacts on Neuronal Excitability.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel plays a critical role in regulating the resting membrane potential and integrating synaptic transmission. Variants of HCN1 have been recognized as causes of epilepsy, and mutant HCN1 channels could act with loss-of-function (LOF), loss- and gain-of-function (LOF and GOF) and gain-of-function (GOF) mechanisms. However, phenotypes and pathogenesis of HCN1-related epilepsy are still poorly understood. This study enrolled five epileptic cases carrying five different HCN1 variants: two pathogenic variants (I380F and S710Rfs*71), two likely pathogenic variants (E240G and A395G), and a paternally inherited variant (V572A). Four variants were novel. Electrophysiological experiments revealed impaired biophysical properties of the identified mutants, including current densities and activation/deactivation kinetics. Moreover, three variants exerted effects on the biophysical properties of wild-type HCN1 channels in heterozygous conditions. Immunofluorescence experiments showed that two variants reduced the protein expression of HCN1channels in neurons. Neurons expressing E240G (GOF) variant showed increased input resistance. However, the variant of I380F (LOF) increased the neuronal firing rate, thus leading to neuronal hyperexcitability. In conclusion, the present study expands the genotypic and phenotypic spectrum of patients with HCN1-related epilepsy and clarifies the underlying mechanisms. We reported five new cases including four unreported likely/pathogenic variants. We provided assessments of biophysical function for each variant, which could help patients to receive individual therapy in the future. We confirmed that HCN1 variants contributed to neuronal hyperexcitability by regulating input resistance and the action potential firing rate, and we have shown that they can affect protein expression in neurons for the first time.

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1).

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure-function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate-gating relationship in TMEM16A remain less explored. Here, we study the gating-permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN- mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN-. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

PATHOGENICITY AND LONG-TERM OUTCOMES OF LIDDLE SYNDROME CAUSED BY A NONSENSE MUTATION OF SCNN1G IN A CHINESE FAMILY

Objective: Liddle syndrome (LS) is a common monogenic hypertension with continuous activation of epithelial sodium channels (ENaCs) encoded by SCNN1A, SCNN1B, and SCNN1G. This study aimed to identify the pathogenicity of a nonsense mutation in SCNN1G in a Chinese family with LS and the outcomes of tailored treatment with amiloride. Design and method: Clinical data were collected from six family members and this mutation site was reported by our team at 2015. To explore the pathogenicity of candidate variants, we constructed mutant and wild-type models in vitro and measured amiloride-sensitive current in Chinese Hamster Ovary cells using patch clamp technique. Participants were followed up for 6 years to monitor their target organ damage after tailored treatment with amiloride. Results: Four family members presented with severe hypertension and hypokalemia, and two had stroke. This nonsense variant resulted in a termination codon at codon 572, truncating the Pro-Pro-Pro-X-Tyr motif. The mutant epithelial sodium channels displayed higher amiloride-sensitive currents than the wild-type channels (P < 0.05). Tailored treatment with amiloride achieved ideal blood pressure control in the patients, and no adverse events occurred during follow-up. Conclusions: We found the pathogenicity of a nonsense SCNN1G mutation (p.Glu571*) with enhanced amiloride-sensitive currents in a LS family. Tailored treatment with amiloride may be an effective strategy for the long-term control of blood pressure and the prevention of cardiovascular events in these patients.

Pathogenicity and Long-Term Outcomes of Liddle Syndrome Caused by a Nonsense Mutation of SCNN1G in a Chinese Family.

ObjectiveLiddle syndrome (LS) is a monogenic hypertension consistent with autosomal dominant inheritance, often with early onset high blood pressure in childhood or adolescence. This study aimed to identify the pathogenicity of a nonsense mutation in SCNN1G in a Chinese family with LS and the long-term outcomes of tailored treatment with amiloride.MethodsTo explore the pathogenicity of candidate variant reported in 2015 by our team, we constructed mutant and wild-type models in vitro and measured amiloride-sensitive current in Chinese Hamster Ovary (CHO) cells using patch clamp technique. Participants were followed up for 7 years after tailored treatment with amiloride.ResultsA nonsense variant was detected in six members, two of whom were pediatric patients. This mutation resulted in a termination codon at codon 572, truncating the Pro-Pro-Pro-X-Tyr motif. The mutant epithelial sodium channels displayed higher amiloride-sensitive currents than the wild-type channels (P < 0.05). Tailored treatment with amiloride achieved ideal blood pressure control in all patients with normal cardiorenal function, and no adverse events occurred during follow-up.ConclusionWe found the pathogenicity of a nonsense SCNN1G mutation (p.Glu571*) with enhanced amiloride-sensitive currents in a LS family with young patients. Tailored treatment with amiloride may be an effective strategy for the long-term control of blood pressure and protection from target organ damage or cardiovascular events, including children and youth patients with LS.

Kir7.1 disease mutant T153I within the inner pore affects K+ conduction.

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.