Voltage Dependence Of Steady-state Activation Research Articles

Functional Characterization of the A414G Loss-of-Function Mutation in HCN4 Associated with Sinus Bradycardia

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p < 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p < 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V

ABSTRACT JZTX-V is a toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. Previous studies had shown that JZTX-V could inhibit the transient outward potassium current of Kv4.2 and Kv4.3 expressed in Xenopus oocytes but had no effects on Kv1.2–1.4. However, the underlying action mechanism of JZTX-V on Kv4.3 remains unclear. In our study, JZTX-V could inhibit not only transient outward potassium currents evoked in small-sized DRG neurons but also Kv4.3-encoded currents expressed in HEK293T cells in the concentration and voltage dependence. The half maximal inhibitory concentration of JZTX-V on Kv4.3 was 9.6 ± 1.2 nM. In addition, the time course for JZTX-V inhibition and release of inhibition after washout were 15.8 ± 1.54 s and 58.8 ± 4.35 s. Electrophysiological assays indicated that 25 nM JZTX-V could shift significantly the voltage dependence of steady-state activation and steady-state inactivation to depolarization. Meanwhile, 25 nM JZTX-V decreased markedly the time constant of activation and inactivation but had no effect on the time constant of recovery from inactivation. To study the molecular determinants of Kv4.3, we performed alanine scanning on a conserved motif of Kv4.3 and assayed the affinity between mutants and JZTX-V. The results not only showed that I273, L275, V283, and F287 were molecular determinants in the conserved motif of Kv4.3 for interacting with JZTX-V but also speculated the underlying action mechanism that the hydrophobic interaction and steric effects played key roles in the binding of JZTX-V with Kv4.3. In summary, our studies have laid a scientific theoretical foundation for further research on the interaction mechanism between JZTX-V and Kv4.3.

Paradoxical effects on voltage-gated Na+conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

A distinct molecular mechanism by which phenytoin rescues a novel long QT 3 variant

BackgroundGenetic variants in SCN5A can result in channelopathies such as the long QT syndrome type 3 (LQT3), but the therapeutic response to Na+ channel blockers can vary. We previously reported a case of an infant with malignant LQT3 and a missense Q1475P SCN5A variant, who was effectively treated with phenytoin, but only partially with mexiletine. Here, we functionally characterized this variant and investigated possible mechanisms for the differential drug actions. MethodsWild-type or mutant Nav1.5 cDNAs were examined in transfected HEK293 cells with patch clamping and biochemical assays. We used computational modeling to provide insights into altered channel kinetics and to predict effects on the action potential. ResultsThe Q1475P variant in Nav1.5 reduced the current density and channel surface expression, characteristic of a trafficking defect. The variant also led to positive shifts in the voltage dependence of steady-state activation and inactivation, faster inactivation and recovery from inactivation, and increased the “late” Na+ current. Simulations of Nav1.5 gating with a 9-state Markov model suggested that transitions from inactivated to closed states were accelerated in Q1475P channels, leading to accumulation of channels in non-inactivated closed states. Simulations with a human ventricular myocyte model predicted action potential prolongation with Q1475P, compared with wild type, channels. Patch clamp data showed that mexiletine and phenytoin similarly rescued some of the gating defects. Chronic incubation with mexiletine, but not phenytoin, rescued the Nav1.5-Q1475P trafficking defect, thus increasing mutant channel expression. ConclusionsThe gain-of-function effects of Nav1.5-Q1475P predominate to cause a malignant long QT phenotype. Phenytoin partially corrects the gating defect without restoring surface expression of the mutant channel, whereas mexiletine restores surface expression of the mutant channel, which may explain the lack of efficacy of mexiletine when compared to phenytoin. Our data makes a case for experimental studies before embarking on a one-for-all therapy of arrhythmias.

SCN9A Epileptic Encephalopathy Mutations Display a Gain-of-function Phenotype and Distinct Sensitivity to Oxcarbazepine.

Genetic mutants of voltage-gated sodium channels (VGSCs) are considered to be responsible for the increasing number of epilepsy syndromes. Previous research has indicated that mutations of one of the VGSC genes, SCN9A (Nav1.7), result in febrile seizures and Dravet syndrome in humans. Despite these recent efforts, the electrophysiological basis of SCN9A mutations remains unclear. Here, we performed a genetic screen of patients with febrile seizures and identified a novel missense mutation of SCN9A (W1150R). Electrophysiological characterization of different SCN9A mutants in HEK293T cells, the previously-reported N641Y and K655R variants, as well as the newly-found W1150R variant, revealed that the current density of the W1150R and N641Y variants was significantly larger than that of the wild-type (WT) channel. The time constants of recovery from fast inactivation of the N641Y and K655R variants were markedly lower than in the WT channel. The W1150R variant caused a negative shift of the G-V curve in the voltage dependence of steady-state activation. All mutants displayed persistent currents larger than the WT channel. In addition, we found that oxcarbazepine (OXC), one of the antiepileptic drugs targeting VGSCs, caused a significant shift to more negative potential for the activation and inactivation in WT and mutant channels. OXC-induced inhibition of currents was weaker in the W1150R variant than in the WT. Furthermore, with administering OXC the time constant of the N641Y variant was longer than those of the other two SCN9A mutants. In all, our results indicated that the point mutation W1150R resulted in a novel gain-of-function variant. These findings indicated that SCN9A mutants contribute to an increase in seizure, and show distinct sensitivity to OXC.

Fluid flow modulates electrical activity in cardiac hERG potassium channels

Fluid movement within the heart generates substantial shear forces, but the effect of this mechanical stress on the electrical activity of the human heart has not been examined. The fast component of the delayed rectifier potassium currents responsible for repolarization of the cardiac action potential, Ikr, is encoded by the human ether-a-go-go related gene (hERG) channel. Here, we exposed hERG1a channel-expressing HEK293T cells to laminar shear stress (LSS) and observed that this mechanical stress increased the whole-cell current by 30-40%. LSS shifted the voltage dependence of steady-state activation of the hERG channel to the hyperpolarizing direction, accelerated the time course of activation and recovery from inactivation, slowed down deactivation, and shifted the steady-state inactivation to the positive direction, all of which favored the hERG open state. In contrast, the time course of inactivation was faster, favoring the closed state. Using specific inhibitors of focal adhesion kinase, a regulator of mechano-transduction via the integrin pathway, we also found that the LSS-induced modulation of the whole-cell current depended on the integrin pathway. The hERG1b channel variant, which lacks the Per-Arnt-Sim (PAS) domain, and long QT syndrome-associated variants having point mutations in the PAS domain were unaffected by LSS, suggesting that the PAS domain in hERG1a channel may be involved in sensing mechanical shear stress. We conclude that a mechano-electric feedback pathway modulates hERG channel activity through the integrin pathway, indicating that mechanical forces in the heart influence its electrical activity.

Transient voltage-activated K+ currents in central antennal lobe neurons: cell type-specific functional properties.

In this study we analyzed transient voltage-activated K+ currents (IA) of projection neurons and local interneurons in the antennal lobe of the cockroach Periplaneta americana The antennal lobe is the first synaptic processing station for olfactory information in insects. Local interneurons are crucial for computing olfactory information and form local synaptic connections exclusively in the antennal lobe, whereas a primary task of the projection neurons is the transfer of preprocessed olfactory information from the antennal lobe to higher order centers in the protocerebrum. The different physiological tasks of these neurons require specialized physiological and morphological neuronal phenotypes. We asked if and how the different physiological phenotypes are reflected in the functional properties of IA, which is crucial for shaping intrinsic electrophysiological properties of neurons. Whole cell patch-clamp recordings from adult male P. americana showed that all their central antennal lobe neurons can generate IA The current exhibited marked cell type-specific differences in voltage dependence of steady-state activation and inactivation, and differences in inactivation kinetics during sustained depolarization. Pharmacological experiments revealed that IA in all neuron types was partially blocked by α-dendrotoxin and phrixotoxin-2, which are considered blockers with specificity for Shaker- and Shal-type channels, respectively. These findings suggest that IA in each cell type is a mixed current generated by channels of both families. The functional role of IA was analyzed in experiments under current clamp, in which portions of IA were blocked by α-dendrotoxin or phrixotoxin-2. These experiments showed that IA contributes significantly to the intrinsic electrophysiological properties, such as the action potential waveform and membrane excitability.NEW & NOTEWORTHY In the insect olfactory system, projection neurons and local interneurons have task-specific electrophysiological and morphological phenotypes. Voltage-activated potassium channels play a crucial role in shaping functional properties of these neurons. This study revealed marked cell type-specific differences in the biophysical properties of transient voltage-activated potassium currents in central antennal lobe neurons.

Exhaustive exercise decreases L-type calcium current by activating endoplasmic reticulum stress.

This study investigated effects of exhaustive exercise on L-type calcium current (ICa,L) and the putative intracellular cascade responsible for the effects. Rats were randomly divided into three treatment groups: sedentary (without exercise), exercised to exhaustion and salubrinal injection before each exhaustive exercise period. Exercise group rats were forced to swim until exhaustion each time for 9 days with 5% body weight attached to the head. Salubrinal (1 mg/kg) or an equivalent volume of placebo solution (dimethyl sulfoxide) was injected via the intraperitoneal route daily for the first 3 days, followed by subcutaneous injections of salubrinal (0.5 mg/kg) or placebo solution daily for 9 days (starting 30 min before exercise). After a 1-day recovery period, whole-cell patch clamping was used to investigate the L-type Ca2+ current (ICa,L), with sedentary control rats. Additionally, endoplasmic reticulum (ER) chaperone protein levels were analyzed. Exhaustive exercise triggered ER stress, demonstrated by elevated expression of ER stress markers: phospho-eIF2α, CCAT/enhancer-binding homologous protein (CHOP) and caspase-12. Compared to controls, ICa,L was inhibited by exhaustive exercise, which was blocked by salubrinal, a selective eIF2α dephosphorylation inhibitor used to inhibit ER stress. These results suggest that ER stress participates in regulation of ICa,L. However, exhaustive exercise did not change the voltage dependence of steady-state activation and inactivation of ICaL, and salubrinal infusion caused no difference in voltage dependence of steady-state activation and inactivation of ICa,L. Exhaustive exercise activates ER stress, thus inhibiting ICaL, which may change the action potential duration and contribute to proarrhythmia.

Calpain inhibitor, MDL 28170 confer electrophysiological, nociceptive and biochemical improvement in diabetic neuropathy

Calpain plays an important role in the pathophysiology of neurological and cardiovascular complications, but its functional association in diabetic neuropathy is not yet elucidated. Therefore, we investigated the role of calpain in modulation of tetrodotoxin-resistant sodium channels (TTX-R Na+ channels) in dorsal root ganglion (DRG) neurons using a pharmacological approach. The effects of a calpain inhibitor, MDL 28170 (3 and 10 mg/kg, i.p.) on TTX-R Na+ channels in DRG neurons of streptozotocin-induced diabetic rats were assessed by using whole-cell patch-clamp technique. In addition to this biochemical, functional and behavioral deficits were also measured. Diabetic rats demonstrated the mechanical allodynia and thermal hyperalgesia with reduced nerve perfusion and conduction velocity as compared to control. MDL 28170 treatments significantly recovered these functional and nociceptive deficits. Moreover, diabetic rats exhibited increased calpain activation, lipid peroxidation and proinflammatory cytokines as compared to control. Drug treatment significantly improved these biochemical deficits. Additionally, DRG neurons from diabetic rats illustrated a significant increase in TTX-R sodium current (INa) density as compared to control. MDL 28170 treatments in diabetic rats significantly blocked the altered channel kinetics with hyperpolarizing shift in voltage-dependence of steady-state activation and inactivation curves. All together, our study provides evidence that calpain activation is directly associated with alterations in TTX-R Na+ channels and triggers functional, nociceptive and biochemical deficits in experimental diabetic neuropathy. The calpain inhibitor, MDL 28710 have shown beneficial effects in alleviating diabetic neuropathy via modulation of TTX-R Na+ channel kinetics and reduction of oxidative stress and neuro-inflammation.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel.

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca(2+)-activated Cl(-) channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl(-) with other anions (PX/PCl) was SCN(-) > I(-) > NO3 (-) > Br(-) > Cl(-) > F(-) > gluconate. When external Cl(-) was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca(2+) were modified according to the sequence of permeability ratios, with anions more permeant than Cl(-) slowing both activation and deactivation and anions less permeant than Cl(-) accelerating them. Moreover, replacement of external Cl(-) with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl(-) with SCN(-) shifted G-V to more negative potentials. Dose-response relationships for Ca(2+) in the presence of different extracellular anions indicated that the apparent affinity for Ca(2+) at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca(2+) in the presence of intracellular SCN(-) also increased compared with that in Cl(-). Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.

Effects of HIV‐1 Tat and morphine on the biophysical properties of sodium channels (653.5)

HIV‐infection continues to be a major clinical manifestation in the combination antiretroviral therapy era (cART), that is enhanced in opioid‐drug abusers. The gastrointestinal (GI) mucosa plays a major role in HIV‐infection as HIV is mostly transmitted across mucosal surfaces. The enteric nervous system (ENS) controls several GI processes including motility and secretion which are specifically affected in HIV‐infected patients. Opioids directly affect the ENS, causing severe constipation. Because the virus does not infect neurons, it is suggested that viral toxins, such as Tat, modulate neuronal function in HIV‐1‐infected patients. Tat has been shown to increase neuronal excitability but not much is known about effects of Tat on enteric neurons in combination with drug abuse. Recent experimental studies in our lab have shown that Tat increases enteric neuronal excitability by shifting the voltage‐dependence of activation of sodium channels to more negative potentials. In contrast, morphine alone decreases the availability of sodium channels in enteric neurons reducing excitability. Voltage gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in neurons. In order to determine the effects of Tat (100 nM) in combination with morphine (0.3µM, 3µM) on the voltage dependence of steady‐state activation/ inactivation of sodium channels, we used a double‐pulse protocol in which a variable conditioning pulse was applied from −100mV to +50mV in 10mV steps for 50 ms followed by a test pulse. In Tat‐treated neurons, the sensitivity to morphine was significantly enhanced. The V0.5 for steady‐state inactivation was shifted in Tat‐treated neurons at 0.3 uM morphine with 50% decrease in maximal availability at ‐100 mV. Morphine did not alter the steady‐state activation. Taken together, data suggest that Tat increases the sensitivity to morphine in enteric neurons that may exacerbate the deleterious effects of morphine on GI motility.Grant Funding Source: Supported by NIDA R01 DA018633, K02 DA027374, K99 DA033878, DK046367.

0265: A novel SCN5A mutation associated with exercise-induced polymorphic ventricular arrhythmias resembling CPVT

Cardiac sodium channel is a complex which includes the poreforming alpha subunit and regulatory proteins that allow sodium influx during the depolarization phase of the ventricular action potential. The alpha subunit, Nav1.5, is encoded by SCN5A . Mutations in this gene are responsible for a wide spectrum of hereditary arrhythmias such as cardiac conduction disease and atrial fibrillation. A mutation in SCN5A , leading to a p.I141V substitution, was identified in a large multigenerational family with an arrhythmia syndrome resembling catecholaminergic polymorphic ventricular tachycardia through whole-exome sequencing. The purpose of this study was to identify the molecular mechanisms linking the p.I141V mutation to this arrhythmia. To evaluate the incidence of this substitution on Nav1.5 function, whole-cell patch-clamp experiments were performed on HEK293 cells transfected with the human alpha and beta subunits. The presence of the p.I141V mutation shifted the voltage-dependence of steady state activation towards more negative potentials. Thus, we studied the effect of this mutation on the sodium window current. Compared to the WT, the p.I141V window current exhibited a larger peak and this peak was shifted towards more negative potentials. To investigate the functional consequences of the cardiac hyperexcitability due to the p.I141V mutation, we incorporated biophysical properties of the mutant into atrial, Purkinje and ventricular cell models. The computational analyses imply that the biophysical changes caused by the p.I141V mutation accelerates the spontaneous rate in the Purkinje cell model, and reduce the excitation threshold leading to an increase of the excitability of the cardiac cells. In conclusion, the present study demonstrates that mutations in SCN5A also may result in exercise-induced polymorphic ventricular premature complexes and tachycardia resembling CPVT.

GW24-e0353 A novel mutation R397Q in KCNQ1 gene caused dilated cardiomyopathy with ventricular tachycardia and prolonged QT interval

ObjectivesDilated cardiomyopathy (DCM) is a major cause of sudden cardiac death with lethal ventricular arrhythmias. So far, the genetic mutations of DCM are mainly related to the sarcomeric protein, and...

GW24-e2441 SCN5A mutation with lethal ventricular arrhythmias and invalidation of lidocaine in acute myocardial infarction

ObjectivesPatients are at high risk for potentially fatal ventricular tachycardia (VT)/ventricular fibrillation (VF) when suffering from acute myocardial infarction (AMI), due to myocardial ischaemia/reperfusion injury or scar formation. Recently, it...

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

Novel Mutations Mapping to the Fourth Sodium Channel Domain of Nav1.7 Result in Variable Clinical Manifestations of Primary Erythromelalgia

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.

Activation Gate Opening Precedes Slow Inactivation in Shaker K+ Channels

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation. Functional and structural studies indicated that channel opening precedes slow inactivation in voltage-gated and KcsA K+ channels, whereas others argued for slow inactivation from the closed sate of the channel as well. None of the previous studies correlated the opening of the of the activation gate (A-gate), formed by the bundle crossing of the S6 segments, with the development of inactivation, which is associated with the structural rearrangement in the selectivity filter. These two gates are coupled and thus, the current experiments addressed the hypothesis that opening of the activation gate must precede slow inactivation.To address this hypothesis we compared the voltage dependence of A-gate opening and that of the development of inactivation in T449A/V474C Shaker-IR channels. Opening of the A-gate was monitored by the accessibility of 474C to Cd2+ from the intracellular side. The membrane potential was changed repeatedly from a holding potential of −120 mV to test potentials ranging from −110 mV to −60 mV in the presence or absence of Cd2+.Our results show that the function describing the voltage dependence of Cd2+ block is shifted toward the negative potentials compared to the voltage dependence of steady-state inactivation curve. This indicates that A-gate opening already occurs at such negative potentials where no inactivation can be detected. Furthermore, the curve representing Cd2+ block is also negative-shifted compared to the voltage dependence of steady-state activation (G-V) curve. This suggests that even at fairly negative holding potentials, at which no macroscopic current can be detected, rare channel openings occur yielding access to the channel cavity. Based on these results we suggest that A-gate opening always precedes structural changes associated with slow inactivation.Supported by OTKA K75904

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

1002 ACUTE INFLAMMATION IN THE PELVIS MODULATES THE EXCITABILITY OF BLADDER EXTRINSIC SENSORY NEURONS VIA ACTIVATION OF VOLTAGE GATED SODIUM CHANNELS AND NON-SELECTIVE CATION CHANNELS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Neurogenic Voiding Dysfunction1 Apr 20101002 ACUTE INFLAMMATION IN THE PELVIS MODULATES THE EXCITABILITY OF BLADDER EXTRINSIC SENSORY NEURONS VIA ACTIVATION OF VOLTAGE GATED SODIUM CHANNELS AND NON-SELECTIVE CATION CHANNELS Qi Lei, Xiao-Qing Pan, Alan Wein, and Anna Malykhina Qi LeiQi Lei More articles by this author , Xiao-Qing PanXiao-Qing Pan More articles by this author , Alan WeinAlan Wein More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chronic pelvic pain is a symptom of many functional pelvic disorders and develops as a result of cross-sensitization in the pelvis. Our recent studies demonstrated increased excitability of convergent dorsal root ganglion neurons receiving input from the distal colon and urinary bladder. The objective of this study was to clarify the mechanisms modulating the expression of voltage gated sodium (Na+) channels in bladder sensory neurons after acute inflammation in the pelvis. METHODS Sprague-Dawley male rats underwent survival surgeries for retrograde labeling of sensory neurons using Fast Blue (1.5 % w/v in water, 20-22 μl). Na+ channels were recorded in lumbosacral (L6-S2) dorsal root ganglia (DRG) neurons isolated from 4 groups of animals: control, resiniferatoxin (RTX) instillation in the colon, trinitrobenzene sulfonic acid (TNBS) induced experimental colitis and double treatment (RTX+TNBS). Animals were sacrificed 3 days after the last treatment. RESULTS Acute colonic inflammation increased the peak amplitude of total Na+ current in bladder DRG neurons by 2-fold at -30 mV (-102.7±41.0 pA/pF in control group vs -229.3±18.1 pA/pF in experimental group, p≤0.05). Activation of TRPV1 receptors in the colon had similar effects on the amplitude of total Na+ current in bladder neurons as in the colitis group. However, colonic application of RTX followed by TNBS-induced colitis significantly enhanced Na+ current in bladder DRG neurons from -102.7±41.0 pA/pF to – 364.6±60.1 pA/pF (n=12, p≤0.05). Acute colitis did not change voltage dependence of steady-state activation nor inactivation in bladder sensory neurons. However, these parameters were affected by RTX treatment. Thus, V1/2 of activation curve of total Na+ current after RTX application had a leftward shift from -27.80±4.78 mV in the control group to -42.20±1.75 mV in RTX group (n=10, p≤0.05) whereas inactivation curve was shifted from -37.75±3.64 mV to -42.94±5.80 mV. Pretreatment with RTX followed by experimental colitis did not modulate the activation kinetics of Na+ current in bladder DRG neurons but caused a leftward shift in inactivation curve from -37.75±3.64 mV to -47.62±1.5 mV (n=11, p≤0.05). CONCLUSIONS Our data provide evidence that acute experimental colitis can modify the expression of Na+ channels in bladder extrinsic sensory neurons via TRPV1 signaling pathways. This data can provide a basis for the development of new therapeutic protocols to treat chronic pelvic pain of genitourinary origin. Glenolden, PA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e389-e390 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qi Lei More articles by this author Xiao-Qing Pan More articles by this author Alan Wein More articles by this author Anna Malykhina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...