Activation Of Voltage-gated Channels Research Articles

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.

Controlling insulin secretion: an exciting TASK.

The -cells of the pancreatic islets of Langerhans secrete insulin in response to metabolic fuels and neurohormonal inputs after a meal. The proper regulation of insulin secretion is critical for the maintenance of glucose homeostasis. An absolute or relative impairment of insulin secretion in the face of insulin resistance underlies the pathophysiology of type 2 diabetes. Accordingly, some of the key antidiabetes agents promote increased insulin secretion from islets, including the newer incretin-based therapeutics and the sulfonylurea class of drugs. The consensus mechanism for glucose-stimulated insulin secretion was elucidated almost 30 years ago and has remained largely unchanged since. A glucose-dependent increase in the intracellular ATP to ADP ratio closes ATP-sensitive potassium (KATP) ion channels (1, 2), themselves the target of the sulfonylurea drugs (3). This results in depolarization of the -cell membrane and the firing of action potentials, which allows the activation of voltage-gated Ca channels and a rise in intracellular Ca that triggers insulin release (4). It is well-recognized, however, that a diverse array of additional ion channels contribute to -cell excitability, and only recently have we begun to understand the roles and molecular identities of the many key channels that shape the electrical response in these cells. Although the outward flux of K mediated by KATP channels dominates the electrical conductance of the -cell membrane in the resting condition,holding themembranepotential of these cells near the equilibrium potential for K ( 70 mV), closure of KATP channels upon glucose stimulation is not in itself sufficient to depolarize the cell. For the -cell to depolarize, an inward depolarizing leak current is required to push the membrane potential toward a more positive equilibrium (or plateau, Figure 1) sufficient to allow the activation of the voltage-gated Na , Ca , and K channels that mediate regenerative action potential firing (5). This depolarizing force is likely mediated by channels that mainly allow the inward leak of Na (and possibly Ca ) such as the transient receptor potential (TRP) family of channels (6) and/or the muscarinic-activated nonselective Na -leak channel (NALNC) (7). Additional currents, such as those mediated by the smallconductance Ca -sensitive K (SK) channels (8) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (9), contribute to the oscillatory nature of the electrical activity that characterizes -cell electrical function. Although required for membrane depolarization after glucose stimulation, these inward leak currents pose a problem of their own: the equilibrium potential for Na and Ca is much too positive ( 50 mV) to allow the rhythmic opening and closing of voltage-dependent ion channels required for action potential firing. What this means is that upon KATP channel closure in response to elevated glucose or sulfonylurea, the inward leak currents could depolarize the -cell too much, unless a counterbalancing outward leak is also present. In the present issue of Endocrinology, Dadi et al (10) present evidence to suggest that this key outward leak current is mediated by TASK-1, a member of the 2-poredomain K (K2P) channel family shown to be expressed at the mRNA level in human -cells only very recently (11, 12) and suggested previously as a target of inhaled anesthetics (13) and for the treatment of multiple sclerosis (14), T cell-mediated autoimmunity (15), and cardiac arrhythmia (16) among other conditions. Using cell-type–specific knockout mice and a selective TASK-1 inhibitor, the authors demonstrate that this channel mediates a back-

Asymmetry in signal propagation between the soma and dendrites plays a key role in determining dendritic excitability in motoneurons.

It is widely recognized that propagation of electrophysiological signals between the soma and dendrites of neurons differs depending on direction, i.e. it is asymmetric. How this asymmetry influences the activation of voltage-gated dendritic channels, and consequent neuronal behavior, remains unclear. Based on the analysis of asymmetry in several types of motoneurons, we extended our previous methodology for reducing a fully reconstructed motoneuron model to a two-compartment representation that preserved asymmetric signal propagation. The reduced models accurately replicated the dendritic excitability and the dynamics of the anatomical model involving a persistent inward current (PIC) dispersed over the dendrites. The relationship between asymmetric signal propagation and dendritic excitability was investigated using the reduced models while varying the asymmetry in signal propagation between the soma and the dendrite with PIC density constant. We found that increases in signal attenuation from soma to dendrites increased the activation threshold of a PIC (hypo-excitability), whereas increases in signal attenuation from dendrites to soma decreased the activation threshold of a PIC (hyper-excitability). These effects were so strong that reversing the asymmetry in the soma-to-dendrite vs. dendrite-to-soma attenuation, reversed the correlation between PIC threshold and distance of this current source from the soma. We propose the tight relation of the asymmetric signal propagation to the input resistance in the dendrites as a mechanism underlying the influence of the asymmetric signal propagation on the dendritic excitability. All these results emphasize the importance of maintaining the physiological asymmetry in dendritic signaling not only for normal function of the cells but also for biophysically realistic simulations of dendritic excitability.

Gene expression patterns in the hippocampus during the development and aging of Glud1(Glutamate Dehydrogenase 1) transgenic and wild type mice

BackgroundExtraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice.ResultsDuring development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young.ConclusionsGlutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases.

Two-Back Makes Step Forward in Brain Imaging Genomics

The combination of brain imaging and genetics promises to identify neural mechanisms underlying complex traits such as cognition and psychiatric disorder. The study by Heck etal. (2014) in this issue of Neuron highlights voltage-gated cation channel activity as a likely link and culprit.

Converging genetic and functional brain imaging evidence links neuronal excitability to working memory, psychiatric disease, and brain activity.

Working memory, the capacity of actively maintaining task-relevant information during a cognitive task, is a heritable trait. Working memory deficits are characteristic for many psychiatric disorders. We performed genome-wide gene set enrichment analyses in multiple independent data sets of young and aged cognitively healthy subjects (n= 2,824) and in a large schizophrenia case-control sample (n= 32,143). The voltage-gated cation channel activity gene set, consisting of genes related to neuronal excitability, was robustly linked to performance in working memory-related tasks across ages and toschizophrenia. Functional brain imaging in 707 healthy participants linked this gene setalso to working memory-related activity in the parietal cortex and the cerebellum. Gene set analyses may help to dissect the molecular underpinnings of cognitive dimensions, brain activity, and psychopathology.

A Quantitative Description of Dendritic Conductances and Its Application to Dendritic Excitation in Layer 5 Pyramidal Neurons

Postsynaptic integration is a complex function of passive membrane properties and nonlinear activation of voltage-gated channels. Some cortical neurons express many voltage-gated channels, with each displaying heterogeneous dendritic conductance gradients. This complexity has hindered the construction of experimentally based mechanistic models of cortical neurons. Here we show that it is possible to overcome this obstacle. We recorded the membrane potential from the soma and apical dendrite of layer 5 (L5) pyramidal neurons of the rat somatosensory cortex. A combined experimental and numerical parameter peeling procedure was implemented to optimize a detailed ionic mechanism for the generation and propagation of dendritic spikes in neocortical L5 pyramidal neurons. In the optimized model, the density of voltage-gated Ca(2+) channels decreased linearly from the soma, and leveled at the distal apical dendrite. The density of the small-conductance Ca(2+)-activated channel decreased along the apical dendrite, whereas the density of the large-conductance Ca(2+)-gated K(+) channel was uniform throughout the apical dendrite. The model predicted an ionic mechanism for the generation of a dendritic spike, the interaction of this spike with the backpropagating action potential, the mechanism responsible for the ability of the proximal apical dendrite to control the coupling between the axon and the dendrite, and the generation of NMDA spikes in the distal apical tuft. Moreover, in addition to faithfully predicting many experimental results recorded from the apical dendrite of L5 pyramidal neurons, the model validates a new methodology for mechanistic modeling of neurons in the CNS.

Genome-wide association study of temperament and tenderness using different Bayesian approaches in a Nellore–Angus crossbred population

Genomic prediction models using Bayesian inference determine marker associations based on available data. The objective of this study was to evaluate marker associations for two traits using different Bayesian models applied to a crossbred population. Nellore–Angus F2, F3 and half-sibling calves were used with records for overall temperament at weaning (TEMP; a subjective scoring system on a 1–9 scale, where 1 is docile or calm and 9 is wild or unruly; n=769) and Warner–Bratzler shear force (WBSF; a measure of tenderness; n=387). After quality control filtering, there were 34,913 SNP markers distributed across the genome available for use (excluding the Y chromosome). An unknown proportion of these markers (designated as π) were assumed not to contribute to the variation in these traits. Bayesian methods employed were BayesC to estimate the ideal π (i.e., value that used as few markers as possible while maintaining heritability, designated as π˜). For WBSF or TEMP, π˜=0.995 or 0.997, respectively. Then BayesB (using π˜) or BayesC (using π=0 or π˜) were fitted to estimate SNP marker effects. Markers were mapped to genes closest to their placement on Bos taurus UMD 3.1 assembly and grouped into 1Mb windows to identify associated regions, where association was determined based on the posterior probability of association of that window being greater than 0.75. No regions associated with either trait were found using π˜, but with π=0, 37 and 147 regions were found to account for more variation than expected under an infinitesimal model for TEMP and WBSF, respectively. Genes from windows identified as associated were used to conduct enrichment analyses. Significant ontology terms related to sodium ion transport and activity, especially voltage-gated channel activity were identified for TEMP, which could be identifying genetic differences between nervous system response to environment and stress stimuli in this population. For WBSF, significant ontology terms related to activity of serine peptidases were identified, but little is known about their true role in muscle tenderness, although they are known to be expressed in muscle.

TRPV1 structures in distinct conformations reveal activation mechanisms

TRP channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (S1-S4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiologic modulation exhibited by TRPV1 and other TRP channels.

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

Robust and high-density biosensors incorporating suspended lipid membranes require microfabricated apertures that can be readily integrated into complex analysis systems. Apertures with well-defined, three-dimensional geometries enable the formation of suspended lipid membranes and facilitate reduced aperture size compared to vertical-walled apertures. Unfortunately, existing methods of producing apertures with well-defined, three-dimensional geometries are based on complex and expensive fabrication procedures, some of which yield apertures in excessively fragile thin-film materials. Here, we describe a microfabrication method utilizing incline and rotate lithography that achieves sloped-wall microapertures in SU-8 polymer substrates with precision control of the aperture diameter, substrate thickness, and wall angle. This approach is simple, is of low cost, and is readily scaled up to allow highly reproducible parallel fabrication. The effect of the incident angle of UV exposure and the size of photomask features on the aperture geometry were investigated, yielding aperture diameters as small as 7 μm and aperture wall angles ranging from 8° to 36° measured from the normal axis. Black lipid membranes were suspended across the apertures and showed normalized conductance values of 0.02-0.05 pS μm(-2) and breakdown voltages of 400-600 mV. The functionality of the resulting sloped-wall microapertures was validated via measurement of reconstituted α-hemolysin activity and the voltage-gated channel activity of alamethicin.

Proton channel HVCN1 is required for effector functions of mouse eosinophils

BackgroundProton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood.ResultsIn the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation.ConclusionsEosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.

Intracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn2+, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2− and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn2+ results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation.Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2− generated within gp91phox in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux.

Time Sensors: Circadian Rhythms in Biologically Closed Electrochemical Circuits of Plants

The circadian clock regulates a wide range of electrophysiological and developmental processes in plants. Here, we discuss the direct influence of a circadian clock on biologically closed electrochemical circuits in vivo. These circuits in the leaves of C. miniata (Kaffir lily), Aloe vera and Mimosa pudica, which regulate their physiology, were analyzed using the charge stimulation method. The electrostimulation was provided with different voltages and electrical charges. Phytosensors memorize daytime and nighttime. Even at continuous light or darkness, plants recognize nighttime or daytime and change the electrical input resistance. The circadian clock can be maintained endogenously and has electrochemical oscillators, which can activate voltage gated ion channels in biologically closed electrochemical circuits. The activation of voltage-gated channels depends on the applied voltage, electrical charge, and the speed of transmission of electrical energy from the electrostimulator to plants.

Structural Basis for Activation of Voltage-Gated Cation Channels

Because of their remarkable roles in electrical cell signaling, voltage-gated cation channels (VGCCs) have been the subject of intense investigations and debate for more than 50 years. Ultimately, the prospective implications of such studies have an impact on our understanding of the molecular properties of VGCCs involved in consciousness, anesthesia, and diseases, to mention a few. The following review aims to summarize our current knowledge of activation of VGCCs by highlighting major methodological innovations in the field and the breakthroughs they allowed. Focusing mainly on insights gained through computer simulations, while acknowledging important experimental findings, we hope to inspire experimentalists to benefit from these approaches in the generation of hypotheses and design of experiments. Also, we outline major future challenges for the field, such as channel modulation, lesser-known receptors, and molecular origins of channel dysfunctions.

Structural Basis for Activation of Voltage-Gated Cation Channels

The critical role of voltage-gated cation channels (VGCCs) relies on a complex voltage-dependent activation mechanism linking two physiologically relevant channel states, open-activated (AT) and closed-resting (RT). Following the early publication of the x-ray crystal structure of the mammalian Kv1.2 channel in the AT conformation, atomistic models for the RT state of the channel have been proposed. For all of these models, structural analyses demonstrated a consensual explanation of experimental data, thereby highlighting the unambiguous nature of these RT structures. Taken together, these structural studies on Kv1.2 have contributed so far with most of our atomic-level knowledge on the activation mechanism of VGCCs. More recently, the x-ray structure of a prokaryotic voltage-gated sodium channel, NavAb, was resolved in a conformation that was interpreted as representative of the pre-open state of the channel. As one of the possible ancestors of the large family of vertebrate voltage-gated Na+ and Ca++ channels, the appearance of the NavAb structure has provided us with a first, and so far unique, template to extend our knowledge towards other members of the large family of VGCCs. Accordingly, in this contribution, we have considered the well-understood AT and RT structures of Kv1.2, equilibrated in a lipid bilayer, as guide structural models to drive a series of molecular dynamics (MD) simulations aimed at to study the activation process of NavAb. While identifying the reported NavAb structure as an intermediate conformation, not fully-activated, our work has enabled us to determine channel conformations likely related to the RT and AT states of the channel. Overall, the structural results support an activation mechanism highly conserved across the entire family of VGCCs.

Theoretical-Experimental Studies of Electric Field-Induced Cell Responses

Tissue injury results in induction of endogenous electric field (EF) that mediates biophysical interactions in the ionic wound environment, which play a crucial role in tissue repair. Externally applied EF may therefore provide a promising therapeutic approach for wound healing; however, information on EF-cell interactions in the wound is limited. This research presents a combined theoretical-experimental approach to study EF interactions with fibroblasts (FBs) in vitro. The cell is modeled as a membrane-enclosed hemisphere surrounded by electrolyte. Maxwell's equations are solved numerically (ANSYS-HFSS) to obtain 3D EF distribution inside and near the cell subjected to an externally applied EF. Experimentally, FBs were exposed to the EF in a parallel-plate capacitor for 72 hours; cell morphology, proliferation and expression of pro-angiogenic growth factors were assessed. Theoretical results demonstrate the spatially-varying EF distribution in the cell membrane, where induced potential may be sufficient to regulate voltage-gated ion channel activation. Cell exposure to the EF resulted in significantly increased expression of placenta growth factor but no change in other parameters. Ongoing studies will determine the role of EF-mediated activation of voltage-gated channels in the cellular response, contributing to development of EF-based therapies for tissue repair.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Models for the Sensitivity of Voltage Gated K Channels to Bilayer Mechanical Stresses

Ion channel Popenvalues measured in membrane patches typically vary with patch history (seal formation mechanics, stretch, time). Pipette aspiration of cell surfaces causes blebbing, an aspiration-intensity dependent dissociation of bilayer and cortical cytoskeleton. This alters the bilayer's mechanical state, rendering it more symmetrical, disordered, fluidized. Once a gigaohm seal forms, adhesion plus aspiration forces contribute to mechanical stress in the bilayer. For voltage-gated channels (VGCs), including Kv channels, Popen(V,t) depends on the bilayer's mechanical state. Aspiration-induced structural changes in the bilayer alter Kv channel Popen both irreversibly and reversibly. Because the specifics of how bilayer mechanics affect VGC activity remain unclear, we have amplified the modeling approaches initiated by Tabarean & Morris 2002 (BJ 82:2982) and by Schmidt et al 2012 (PNAS 109:10352) for Shaker and other Kv channels. The Markov models considered are variations on kinetic schemes introduced by Aldrich & colleagues (Zagotta et al 1994 JGP 103:321). In some versions we consider (non-N-type) inactivation and late concerted transitions. We tested model variations for their ability to describe voltage clamp responses of Kv1 Shaker (WT, 5aa, ILT) and Kv3 Shaw without/with pipette aspiration. The main question is how do the features of I(V,t) and Q(V,t) families vary under mechanical stress? We assign mechanosensitivity to one or several of the voltage-dependent transitions and the pore opening/closing transition and show how time courses and I(V) and Q(V) change. A stimulus “opposite” to bilayer stretch, in that it increases lipid packing order and bilayer thickness, is hyperbaric pressure. These stimuli, more accurately, fall along a bilayer-mechanical continuum. Since ocean-dwelling organisms from bacteria to cetaceans routinely experience hyperbaric pressures, we extended our analysis to explore, for selected models, how hyperbaric pressures might affect Kv gating. ∗Funded by NSERC (Canada).

Activity-Dependent Alternative Splicing Increases Persistent Sodium Current and Promotes Seizure

Activity of voltage-gated Na channels (Na(v)) is modified by alternative splicing. However, whether altered splicing of human Na(v)s contributes to epilepsy remains to be conclusively shown. We show here that altered splicing of the Drosophila Na(v) (paralytic, DmNa(v)) contributes to seizure-like behavior in identified seizure mutants. We focus attention on a pair of mutually exclusive alternate exons (termed K and L), which form part of the voltage sensor (S4) in domain III of the expressed channel. The presence of exon L results in a large, non-inactivating, persistent I(Nap). Many forms of human epilepsy are associated with an increase in this current. In wild-type (WT) Drosophila larvae, ∼70-80% of DmNa(v) transcripts contain exon L, and the remainder contain exon K. Splicing of DmNa(v) to include exon L is increased to ∼100% in both the slamdance and easily-shocked seizure mutants. This change to splicing is prevented by reducing synaptic activity levels through exposure to the antiepileptic phenytoin or the inhibitory transmitter GABA. Conversely, enhancing synaptic activity in WT, by feeding of picrotoxin is sufficient to increase I(Nap) and promote seizure through increased inclusion of exon L to 100%. We also show that the underlying activity-dependent mechanism requires the presence of Pasilla, an RNA-binding protein. Finally, we use computational modeling to show that increasing I(Nap) is sufficient to potentiate membrane excitability consistent with a seizure phenotype. Thus, increased synaptic excitation favors inclusion of exon L, which, in turn, further increases neuronal excitability. Thus, at least in Drosophila, this self-reinforcing cycle may promote the incidence of seizure.

Using Voltage-Clamp Fluorometry Technique to Study the Mechanism of the Cooperativity Between Hv Channel Subunits

In the immune system, the activity of voltage-gated proton channels has been shown to play a key role in the charge compensation for the electron extrusion by NADPH oxidase during respiratory burst in phagocytes. In addition, this channel was also found in many other cell types including lung airway epithelia cells, sperms and high metastatic breast cancer cells, in which they have been implicated in the phathology of asthma, pH dependent spermatozoa and tumor metastasis, respectively. It was not until 2006 that two independent groups discovered the genes coding for the voltage-gated proton (Hv) channels. It was shown that the Hv channel is a dimer and that there is a strong coopertivity between two subunits during activation of this channel. However, how the two subunits in Hv channels cooperatively activate the channel and to what extent this coopertivity affect the channel activation are unknown. In this study, we investigate the detailed molecular mechanism of coopertivity in the dimeric Hv channel by using the combination of two electrodes voltage clamp (TEVC) and voltage clamp fluorometry (VCF). We measure the voltage sensor movement and pore opening of each subunit in a linked-dimer that has two subunits with different activation voltage ranges. Thereby we can determine how one subunit affects the other subunit during voltage sensor movement and pore opening.