Activation Of Voltage-gated Channels Research Articles

A novel variant c.902C>A (p. A301D) in KCNQ4 associated with non-syndromic deafness 2A in a Chinese family.

Deafness autosomal dominant 2A (DFNA2A) is related to non-syndromic genetic hearing impairment. The KCNQ4 (Potassium Voltage-Gated Channel Subfamily Q Member 4) can lead to DFNA2A. In this study, we report a case of autosomal dominant non-syndromic hearing loss with six family members as caused by a novel variant in the KCNQ4 gene. The whole-exome sequencing (WES) and pure tone audiometry were performed on the proband of the family. Sanger sequencing was conducted on family members to determine if the novel variant in the KCNQ4 gene was present. Evolutionary conservation analysis and computational tertiary structure protein prediction of the wild-type KCNQ4 protein and its variant were then performed. In addition, voltage-gated channel activity of the wild-type KCNQ4 protein and its variant were tested using whole-cell patch clamp. It was observed that the proband had inherited autosomal dominant, non-syndromic sensorineural hearing loss as a trait. A novel co-segregating heterozygous missense variant (c.902C>A, p.Ala301Asp) of the KCNQ4 gene was identified in the proband and other five affected family members. This variant was predicted to cause an alanine-to-aspartic acid substitution at position 301 in the KCNQ4 protein. The alanine at position 301 is well conserved across different species. Whole-cell patch clamp showed that there was a significant difference between the WT protein currents and the mutant protein currents in the voltage-gated channel activity. In the present study, performing WES in conjunction with Sanger sequencing enhanced the detection of a novel, potentially causative variant (c301 A>G; p.Ala301Asp) in exon 6 of the KCNQ4 gene. Therefore, our findings contributed to the mutation spectrum of the KCNQ4 gene and may be useful in the diagnosis and gene therapy of deafness autosomal dominant 2A.

Voltage-gated potassium channels control extended access cocaine seeking: a role for nucleus accumbens astrocytes.

Dopaminergic signaling in the nucleus accumbens shell (NAc) regulates neuronal activity relevant to reward-related learning, including cocaine-associated behaviors. Although astrocytes respond to dopamine and cocaine with structural changes, the impact of dopamine and cocaine on astrocyte functional plasticity has not been widely studied. Specifically, behavioral implications of voltage-gated channel activity in the canonically non-excitable astrocytes are not known. We characterized potassium channel function in NAc astrocytes following exposure to exogenous dopamine or cocaine self-administration training under short (2 h/day) and extended (6 h/day) access schedules. Electrophysiological, Ca2+ imaging, mRNA, and mass spectrometry tools were used for molecular characterization. Behavioral effects were examined after NAc-targeted microinjections of channel antagonists and astroglial toxins. Exogenous dopamine increased activity of currents mediated by voltage-gated (Kv7) channels in NAc astrocytes. This was associated with a ~5-fold increase in expression of Kcnq2 transcript level in homogenized NAc micropunches. Matrix-assisted laser desorption/ionization mass spectrometry revealed increased NAc dopamine levels in extended access, relative to short access, rats. Kv7 inhibition selectively increased frequency and amplitude of astrocyte intracellular Ca2+ transients in NAc of extended access rats. Inhibition of Kv7 channels in the NAc attenuated cocaine-seeking in extended access rats only, an effect that was occluded by microinjection of the astrocyte metabolic poison, fluorocitrate. These results suggest that voltage-gated K+ channel signaling in NAc astrocytes is behaviorally relevant, support Kv7-mediated regulation of astrocyte Ca2+ signals, and propose novel mechanisms of neuroglial interactions relevant to drug use.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.

Abstract P1146: Global Analysis Of Murine Cardiac Gene Program Regulated By Thyroid Hormone And Transcriptional Repressor Rest

Background: During adult heart failure, transcriptional reprogramming of fetal cardiac genes coincides with dysregulated heart functions. However, the underlying mechanisms remain incompletely understood. Thyroid hormone and RE1 silencing transcription factor (REST) are known regulators of cardiac gene reprogramming. Here, we report a global analysis of their co-regulatory functions. Methods: We generated a dual Myh6 and Myh7 mouse reporter line. This line was crossed with a conditional Rest allele to generate myocardial Rest knockout mice. Control and Rest null mice were treated with propylthiouracil (PTU) to inhibit thyroid hormone synthesis. PTU treatment induced heart failure and adult-to-fetal cardiac gene remodeling. Single nuclei multiomic profiling was performed, linking gene expression and gene regulatory network information from each nuclei to elucidate the impact of PTU or REST or both on cardiac gene reprogramming. Samples were collected to validate the newly identified factors involved in reprogramming. Results: Data analysis showed that PTU treatment upregulates transcription factor binding involving circadian gene expression ( Cry2 , Per2 , Per3 ). Downregulated genes are related to voltage-gated channel activity and BMP/TGFB activities involving heart development ( Myh6 , Nrg1 , Bmpr1a ) and SA node action potential ( Scn5a , Hcn2 , Ryr2 ). In contrast, Rest knockout upregulated genes are associated with arrhythmia, i.e. , cAMP catabolism ( Pde4d , Pde7a , Pde7b ), heart conduction ( Cacna1c , Gja1 , Pln ), and ion transport ( Kcnip2 , Pde4d, Esr1 ). Downregulated genes are involved in protein functions involving integrin-mediated signaling ( Itgal , Itga4 , Itga5 ), cytoskeleton organization ( Ctnna2 , Actb , Xirp1 ), and fiber organization ( Tgfb3 , Smad3 , Ptk2b ). Furthermore, a subset of genes was found to be co-regulated by thyroid hormone and REST, including Ccn2 , Myh9 , Nmrk2 , Fgr , Itga4 , Prkcb , Cspg4 which are involved in integrin signaling pathway, immune activation, and angiogenesis. Conclusion: Thyroid hormone and REST have shared function in cardiac gene reprogramming involving cardiac conduction and fibrosis as well as their own newly discovered, unique roles regulating circadian genes and cAMP catabolism, respectively.

Secondhand Smoke Decreased Excitability and Altered Action Potential Characteristics of Cardiac Vagal Neurons in Mice.

Background: Secondhand smoke (SHS), a major indoor pollutant, is a significant risk factor for cardiovascular morbidity and mortality including arrhythmias and sudden cardiac death. Exposure to SHS can produce autonomic imbalance, as evidenced by reduced heart rate variability (HRV)—a clinical metric of cardiac vagal regulation. Currently, the mechanisms through which SHS changes the vagal preganglionic neuronal inputs to the heart to produce this remains unknown.Objectives: To characterize the effect of SHS on both the excitability and action potential (AP) characteristics of anatomically identified cardiac vagal neurons (CVNs) in the nucleus ambiguus and examine whether SHS alters small conductance calcium-activated potassium (SK) channel activity of these CVNs.Methods: Adult male mice were exposed to four weeks of filtered air or SHS (3 mg/m3) 6 h/day, 5 day/week. Using patch-clamp recordings on identified CVNs in brainstem slices, we determined neuronal excitability and AP characteristics with depolarizing step- and ramp-current injections.Results: Four weeks of SHS exposure reduced spiking responses to depolarizing current injections and increased AP voltage threshold in CVNs. Perfusion with apamin (20 nM) magnified these SHS-induced effects, suggesting reduced SK channel activity may serve to minimize the SHS-induced decreases in CVNs excitability. Medium afterhyperpolarization (a measurement of SK channel activity) was smaller in the SHS group, further supporting a lower SK channel activity. AP amplitude, rise rate, fast afterhyperpolarization amplitude (a measurement of voltage-gated channel activity), and decay rate were higher in the SHS group at membrane voltages more positive to 0 mV, suggesting altered inactivation properties of voltage-dependent channels underlying APs.Discussion: SHS exposure reduced neuronal excitability of CVNs with compensatory attenuation of SK channel activity and altered AP characteristics. Neuroplasticity of CVNs could blunt regulatory cardiac vagal signaling and contribute to the cardiovascular consequences associated with SHS exposure, including reduced HRV.

Developmental Profile of Psychiatric Risk Associated With Voltage-Gated Cation Channel Activity

BackgroundRecent breakthroughs in psychiatric genetics have implicated biological pathways onto which genetic risk for psychiatric disorders converges. However, these studies do not reveal the developmental time point(s) at which these pathways are relevant. MethodsWe aimed to determine the relationship between psychiatric risk and developmental gene expression relating to discrete biological pathways. We used postmortem RNA sequencing data (BrainSeq and BrainSpan) from brain tissue at multiple prenatal and postnatal time points, with summary statistics from recent genome-wide association studies of schizophrenia, bipolar disorder, and major depressive disorder. We prioritized gene sets for overall enrichment of association with each disorder and then tested the relationship between the association of their constituent genes with their relative expression at each developmental stage. ResultsWe observed relationships between the expression of genes involved in voltage-gated cation channel activity during early midfetal, adolescence, and early adulthood time points and association with schizophrenia and bipolar disorder, such that genes more strongly associated with these disorders had relatively low expression during early midfetal development and higher expression during adolescence and early adulthood. The relationship with schizophrenia was strongest for the subset of genes related to calcium channel activity, while for bipolar disorder, the relationship was distributed between calcium and potassium channel activity genes. ConclusionsOur results indicate periods during development when biological pathways related to the activity of calcium and potassium channels may be most vulnerable to the effects of genetic variants conferring risk for psychiatric disorders. Furthermore, they indicate key time points and potential targets for disorder-specific therapeutic interventions.

Hysteretic Behavior in Voltage-Gated Channels.

An ever-growing body of evidence has shown that voltage-gated ion channels are likely molecular systems that display hysteresis in their activity. This phenomenon manifests in the form of dynamic changes in both their voltage dependence of activity and their deactivation kinetics. The goal of this review is to provide a clear definition of hysteresis in terms of the behavior of voltage-gated channels. This review will discuss the basic behavior of voltage-gated channel activity and how they make these proteins into systems displaying hysteresis. It will also provide a perspective on putative mechanisms underlying hysteresis and explain its potential physiological relevance. It is uncertain whether all channels display hysteresis in their behavior. However, the suggested notion that ion channels are hysteretic systems directly collides with the well-accepted notion that ion channel activity is stochastic. This is because hysteretic systems are regarded to have “memory” of previous events while stochastic processes are regarded as “memoryless.” This review will address this apparent contradiction, providing arguments for the existence of processes that can be simultaneously hysteretic and stochastic.

Role of the lncRNA BACE1-AS in shared disease mechanisms between heart failure and Alzheimer's disease

Abstract Background BACE1 encodes for β-secretase, the key enzyme involved in β-amyloid (βA) generation, a peptide well known for its involvement in Alzheimer's disease (AD). Of note, heart failure (HF) and AD share several risk factors and effectors. We recently showed that, in the heart of ischemic HF patients, the levels of both BACE1, its antisense RNA BACE1-AS and βA are all increased. BACE1-AS positively regulates the expression of BACE1, triggering βA intracellular accumulation, and its overexpression or βA administration induce cardiovascular-cell apoptosis. Aim To characterize the transcripts of the BACE1 locus and to investigate the molecular mechanisms underpinning BACE1-AS regulation of cell vitality. Methods By PCR and sequencing, we studied in the heart the expression of a variety of antisense BACE1 transcripts predicted by FANTOM CAT Epigenome. We studied BACE1 RNA stability by BrdU pulse chase experiments (BRIC assay). The cellular localization of BACE1-AS RNA was investigated by in situ hybridization assay. BACE1-AS binding RNAs were evaluated by BACE1-AS-MS2-Tag pull-down in AC16 cardiomyocytes followed by RNA-seq. Enriched RNAs were validated by qPCR and analysed by bioinformatics comparison with publicly available gene expression datasets of AD brains. Results We readily detected several antisense BACE1 transcripts expressed in AC16 cardiomyocytes; however, only BACE1-AS RNAs overlapping exon 6 of BACE1 positively regulated BACE1 mRNA levels, acting by increasing its stability. BACE1 silencing reverted cell apoptosis induced by BACE1-AS expression, indicating that BACE1 is a functional target of BACE1-AS. However, in situ hybridization experiments indicated a mainly nuclear localization for BACE1-AS, which displayed a punctuated distribution, compatible with chromatin association and indicative of potential additional targets. To identify other BACE1-AS binding RNAs, a BACE1-AS-MS2-tag pull-down was performed and RNA-seq of the enriched RNAs identified 698 BACE1-AS interacting RNAs in cardiomyocytes. Gene ontology of the BACE1-AS binding RNAs identified categories of relevance for cardiovascular or neurological diseases, such as dopaminergic synapse, glutamatergic synapse, calcium signalling pathway and voltage-gated channel activity. In spite of the differences between brain and heart transcriptomes, BACE1-AS-interacting RNAs identified in cardiomyocytes were significantly enriched in transcripts differentially expressed in AD brains as well as in RNAs expressed by enhancer genomic regions that are significantly hypomethylated in AD brains. Conclusions These data shed a new light on the complexity of BACE1-AS locus and on the existence of RNAs interacting with BACE1-AS with a potential as enhancer-RNAs. Moreover, the dysregulation of the BACE1-AS/BACE1/βA pathway may be a common disease mechanism shared by cardiovascular and neurological degenerative diseases. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Health Ministery_Ricerca Corrente 2020

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.

Weighted gene co-expression network analysis of chronic kidney disease and hemodialysis patients

The group of heterogeneous disorders affecting the kidneys' structure and function is generally known as chronic kidney disease (CKD). In developed countries, most patients at the terminal CKD stage are treated with hemodialysis in order to increase their lifespan. Here, we used a microarray data set of patients with CKD, patients on hemodialysis, and healthy controls along with a weighted gene co-expression network analysis (WGCNA). We detected seven modules by using the WGCNA, and the blue, turquoise, and brown modules were highly enriched with differentially expressed genes (DEGs). Blue, turquoise, and brown module genes were the most significantly enriched in the gene ontology (GO) terms involved in oxidative phosphorylation, voltage-gated cation channel activity, and regulation of striated muscle cell differentiation, respectively. The brown module also included several GO terms involved in immunity. The turquoise module contained many genes that are known to cause neurological and heart diseases due to decreased expression. The brown module contained recombination signal binding protein for immunoglobulin κ J region (RBPJ) genes that are involved in vascular homeostasis and the survival of memory CD4-positive T cells. The decreased expression of RBPJ may be related to cardiovascular complications and susceptibility to infection. These findings contribute to a more complete understanding of the pathology of CKD and hemodialysis patients and complications including cardiovascular events and infectious diseases.

Serotonin, norepinephrine, and acetylcholine differentially affect astrocytic potassium clearance to modulate somatosensory signaling in male mice.

Changes in extracellular potassium ([K+ ]e ) modulate neuronal networks via changes in membrane potential, voltage-gated channel activity, and alteration to transmission at the synapse. Given the limited extracellular space in the central nervous system, potassium clearance is crucial. As activity-induced potassium transients are rapidly managed by astrocytic Kir4.1 and astrocyte-specific Na+ /K+ -ATPase, any neurotransmitter/neuromodulator that can regulate their function may have indirect influence on network activity. Neuromodulators differentially affect cortical/thalamic networks to align sensory processing with differing behavioral states. Given serotonin (5HT), norepinephrine (NE), and acetylcholine (ACh) differentially affect spike frequency adaptation and signal fidelity ("signal-to-noise") in somatosensory cortex, we hypothesize that [K+ ]e may be differentially regulated by the different neuromodulators to exert their individual effects on network function. This study aimed to compare effects of individually applied 5HT, NE, and ACh on regulating [K+ ]e in connection to effects on cortical-evoked response amplitude and adaptation in male mice. Using extracellular field and K+ ion-selective recordings of somatosensory stimulation, we found that differential effects of 5HT, NE, and ACh on [K+ ]e regulation mirrored differential effects on amplitude and adaptation. 5HT effects on transient K+ recovery, adaptation, and field post-synaptic potential amplitude were disrupted by barium (200µM), whereas NE and ACh effects were disrupted by ouabain (1µM) or iodoacetate (100µM). Considering the impact [K+ ]e can have on many network functions; it seems highly efficient that neuromodulators regulate [K+ ]e to exert their many effects. This study provides functional significance for astrocyte-mediated buffering of [K+ ]e in neuromodulator-mediated shaping of cortical network activity.

Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90-231 Neurotoxic Effects in Cerebellar Granule Neurons.

Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrPSc). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca++] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca++ entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca++] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca++] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca++] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE2 release from glial cells. Thus, Ca++ homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides induce neurotoxic signals via both direct neuron interaction and glia activation through different mechanisms responsible of calcium homeostasis disruption in neurons and potentiating each other: the activation of excitotoxic pathways via NMDA receptors and the release of radical species that establish an oxidative milieu.

Effects of a GABAC Receptor Blocker on the Formation of Evoked Potentials in Neuronal Columns in the Rat Somatosensory Cortex

Microelectrode studies of the evoked activity of neuronal columns in the barrel cortex in rats showed that the GABAC receptor blocker 1,2,5,6-tetrahydropyridin-4-ylmethylphosphonic acid (TPMPA) had an activatory effect on the secondary components of evoked potentials (EP) mainly of the upper afferent modules of columns as compared with the lower efferent layers. This is evidence that GABAC receptors are located on the presynaptic terminals of thalamocortical glutamatergic afferents and ascending apical dendrites of pyramidal cells. Blockade of GABAC influences with TPMPA led to dose-dependent depolarization of afferents with development of presynaptic inhibition and suppression of the primary responses (PR) of EP. The different effects of the GABAC receptor blocker on the secondary components of EP in the upper layers of the cortex are due to the development of afterhyperpolarization of neurons after high-amplitude PR and afterdepolarization after low-amplitude responses, with subsequent activation of different voltage-gated channels and the formation of different gradients in the direct current (DC) potential of the cortex.

Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response.

Cellular microenvironment modulates the galvanotaxis of brain tumor initiating cells.

Galvanotaxis is a complex process that represents the collective outcome of various contributing mechanisms, including asymmetric ion influxes, preferential activation of voltage-gated channels, and electrophoretic redistribution of membrane components. While a large number of studies have focused on various up- and downstream signaling pathways, little is known about how the surrounding microenvironment may interact and contribute to the directional response. Using a customized galvanotaxis chip capable of carrying out experiments in both two- and three-dimensional microenvironments, we show that cell-extracellular matrix (ECM) interactions modulate the galvanotaxis of brain tumor initiating cells (BTICs). Five different BTICs across three different glioblastoma subtypes were examined and shown to all migrate toward the anode in the presence of a direct-current electric field (dcEF) when cultured on a poly-L-ornithine/laminin coated surface, while the fetal-derived neural progenitor cells (fNPCs) migrated toward the cathode. Interestingly, when embedded in a 3D ECM composed of hyaluronic acid and collagen, BTICs exhibited opposite directional response and migrated toward the cathode. Pharmacological inhibition against a panel of key molecules involved in galvanotaxis further revealed the mechanistic differences between 2- and 3D galvanotaxis in BTICs. Both myosin II and phosphoinositide 3-kinase (PI3K) were found to hold strikingly different roles in different microenvironments.

Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop.

Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.

Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine.

Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine.

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca2+ channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca2+ channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca2+ channels (CaV1.3 and CaV1.2) but not the N-type Ca2+ channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca2+ signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca2+ channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca2+ channel activation.

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.