A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis

Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels.     

Leaky channels make weak muscles

Mutations in the skeletal muscle voltage-gated calcium channel (Ca_V1.1) have been associated with hypokalemic periodic paralysis, but how the pathogenesis of this disorder relates to the functional consequences of mutations was unclear. In this issue of the JCI, Wu and colleagues recapitulate the disease by generating a novel knock-in Ca_V1.1 mutant mouse and use this model to investigate the cellular and molecular features of pathogenesis. They demonstrated an aberrant muscle cell current conducted through the Ca_V1.1 voltage-sensor domain (gating pore current) that explains an abnormally depolarized muscle membrane and the failure of muscle action potential firing during challenge with agents known to provoke periodic paralysis. Their work advances understanding of molecular and cellular mechanisms underlying an inherited channelopathy. Ion channels are ubiquitous membrane proteins that confer selective ionic permeability to the plasmalemma or intracellular membranes and enable a wide variety of important physiological processes, including membrane excitability, synaptic transmission, signal transduction, cell volume regulation, and transcellular ion transport. The vital nature of ion channels is reflected by the existence of inherited disorders caused by mutations in genes that encode these proteins (1–5). These “channelopathies” represent more than 50 human genetic diseases, including several affecting skeletal muscle contraction, such as the periodic paralyses and nondystrophic myotonias (see “Muscle channelopathies”).     

Epilepsy channelopathies go neddy: stabilizing NaV1.1 channels by neddylation

Loss-of-function mutations of SCN1A encoding the pore-forming α subunit of the Na_V1.1 neuronal sodium channel cause a severe developmental epileptic encephalopathy, Dravet syndrome (DS). In this issue of the JCI, Chen, Luo, Gao, et al. describe a phenocopy for DS in mice deficient for posttranslational conjugation with neural precursor cell expressed, developmentally downregulated 8 (NEDD8) (neddylation), selectively engineered in inhibitory interneurons. Pursuing the possibility that this phenotype is also caused by loss of Na_V1.1, Chen, Luo, Gao, and colleagues show that interneuron excitability and GABA release are impaired, Na_V1.1 degradation rate is increased with a commensurate decrease of Na_V1.1 protein, and Na_V1.1 is a substrate for neddylation. These findings establish neddylation as a mechanism for stabilizing Na_V1.1 subunits and suggest another pathomechanism for epileptic sodium channelopathy.     

Neddylation stabilizes Nav1.1 to maintain interneuron excitability and prevent seizures in murine epilepsy models

The excitability of interneurons requires Na_v1.1, the α subunit of the voltage-gated sodium channel. Na_v1.1 deficiency and mutations reduce interneuron excitability, a major pathological mechanism for epilepsy syndromes. However, the regulatory mechanisms of Na_v1.1 expression remain unclear. Here, we provide evidence that neddylation is critical to Na_v1.1 stability. Mutant mice lacking Nae1, an obligatory component of the E1 ligase for neddylation, in parvalbumin-positive interneurons (PVINs) exhibited spontaneous epileptic seizures and premature death. Electrophysiological studies indicate that Nae1 deletion reduced PVIN excitability and GABA release and consequently increased the network excitability of pyramidal neurons (PyNs). Further analysis revealed a reduction in sodium-current density, not a change in channel property, in mutant PVINs and decreased Na_v1.1 protein levels. These results suggest that insufficient neddylation in PVINs reduces Na_v1.1 stability and thus the excitability of PVINs; the ensuing increased PyN activity causes seizures in mice. Consistently, Na_v1.1 was found reduced by proteomic analysis that revealed abnormality in synapses and metabolic pathways. Our findings describe a role of neddylation in maintaining Na_v1.1 stability for PVIN excitability and reveal what we believe is a new mechanism in the pathogenesis of epilepsy.     

Initiation of migraine-related cortical spreading depolarization by hyperactivity of GABAergic neurons and NaV1.1 channels

Spreading depolarizations (SDs) are involved in migraine, epilepsy, stroke, traumatic brain injury, and subarachnoid hemorrhage. However, the cellular origin and specific differential mechanisms are not clear. Increased glutamatergic activity is thought to be the key factor for generating cortical spreading depression (CSD), a pathological mechanism of migraine. Here, we show that acute pharmacological activation of Na_V1.1 (the main Na⁺ channel of interneurons) or optogenetic-induced hyperactivity of GABAergic interneurons is sufficient to ignite CSD in the neocortex by spiking-generated extracellular K⁺ build-up. Neither GABAergic nor glutamatergic synaptic transmission were required for CSD initiation. CSD was not generated in other brain areas, suggesting that this is a neocortex-specific mechanism of CSD initiation. Gain-of-function mutations of Na_V1.1 (SCN1A) cause familial hemiplegic migraine type-3 (FHM3), a subtype of migraine with aura, of which CSD is the neurophysiological correlate. Our results provide the mechanism linking Na_V1.1 gain of function to CSD generation in FHM3. Thus, we reveal the key role of hyperactivity of GABAergic interneurons in a mechanism of CSD initiation, which is relevant as a pathological mechanism of Na_v1.1 FHM3 mutations, and possibly also for other types of migraine and diseases in which SDs are involved.     

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (Na_V1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased Na_V1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting Na_V1.7 alpha subunit mRNA attenuated the expression of Na_V1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective Na_V1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the Na_V1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances Na_V1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of Na_V1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that Na_V1.7 is a target for the treatment of chronic muscle pain.PerspectiveWe demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of Na_V1.7 in nociceptors.     

A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels,Ca2+ Signaling,and Gene Expression in T Lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K⁺ channels (K_V and K_Ca, respectively), resulting in depolarization of the membrane potential. As a result, Ca²⁺ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca²⁺ signals after thapsigargin stimulation, as well as oscillatory Ca²⁺ signals, but not the Ca²⁺ transient after TCR stimulation. K⁺ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked K_V and K_Ca channels. Progesterone effectively blocked a broad spectrum of K⁺ channels, reducing both Kv1.3 and charybdotoxin–resistant components of K_V current and K_Ca current in T cells, as well as blocking several cloned K_V channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na⁺ channel, an inward rectifier K⁺ channel, or on lymphocyte Ca²⁺ and Cl⁻ channels. We propose that direct inhibition of K⁺ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.     

Hyperexcitable interneurons trigger cortical spreading depression in an Scn1a migraine model

Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na⁺ channel Na_V1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1a^L1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1a^L1649Q knock-in mice compared with WT mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na⁺-channel inactivation and increased ramp Na⁺ currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K⁺-sensitive electrodes revealed an increase in extracellular K⁺ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1a^L1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na⁺ currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.     

A βIV-spectrin/CaMKII signaling complex is essential for membrane excitability in mice

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na⁺ channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na⁺ channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β_IV-spectrin as a multifunctional regulatory platform for Na⁺ channels in mice. We found that β_IV-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β_IV-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na⁺ channels, via direct phosphorylation by β_IV-spectrin–targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.     

Location,location, regulation: a novel role for β-spectrin in the heart

Voltage-gated Na⁺ channels (VGSCs) are responsible for the rising phase of the action potential in excitable cells, including neurons and skeletal and cardiac myocytes. Small alterations in gating properties can lead to severe changes in cellular excitability, as evidenced by the plethora of heritable conditions attributed to mutations in VGSCs highlighting the need to better understand VGSC regulation. In this issue of the JCI, Hund et al. identify the ability of a key structural protein, β_IV-spectrin, to bind and recruit Ca²⁺/calmodulin kinase II to the channel at a cellular location key to successful action potential initiation and propagation, where it can mediate function and excitability.     

Changes in the expression of NaV1.7, NaV1.8 and NaV1.9 in a distinct population of dorsal root ganglia innervating the rat knee joint in a model of chronic inflammatory joint pain

Voltage‐gated sodium channels play an essential role in regulating the excitability of nociceptive primary afferent neurones. In particular the tetrodotoxin‐sensitive (TTX‐S) Na_V1.7 and the tetrodotoxin‐resistant (TTX‐R) Na_V1.8 and Na_V1.9 channels have been suggested to play a role in inflammatory pain. Previous work has revealed acute administration of inflammatory mediators, such as Freund's complete adjuvant (FCA) or carrageenan caused an upregulation in the levels of Na_V1.7 and Na_V1.8 protein in DRG (dorsal root ganglia) tissue up to 4 days post‐insult. In the present study, the expression of Na_V1.7, Na_V1.8 and Na_V1.9 was examined over a 28 day timecourse during a rat model of FCA‐induced chronic inflammatory joint pain. Using the retrograde tracer Fast Blue (FB) and specific Na_V1.7, Na_V1.8 and Na_V1.9 sodium channel antibodies, immunohistochemical staining techniques were used to study sodium channel expression in a distinct population of L3–L5 knee joint afferent DRGs. In the ganglia, counts were made of positively labelled cells in the FB population. The results demonstrate that, following FCA injection, Na_V1.9 expression is upregulated at days 14, 21 and 28 post‐FCA, with Na_V1.7 and Na_V1.8 showing increased channel expression at days 14 and 28. These observations are accompanied by a unilateral joint hypersensitivity in the FCA‐injected knee indicated by a behavioural shift in weight distribution measured using an incapacitance tester. The increased presence of these channels suggests that Na_V1.7, Na_V1.8 and Na_V1.9 play a role, at least in part, in the maintenance of chronic inflammatory pain several weeks after the initial insult.     

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca²⁺ channel Ca_V1.2 in nonexcitable cells. How abnormal Ca²⁺ influx through Ca_V1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established Ca_V1.2 roles. Here, we show that Ca_V1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca²⁺ influx through Ca_V1.2 regulates jaw development. Cranial neural crest migration was unaffected by Ca_V1.2 knockdown, suggesting a role for Ca_V1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca²⁺ signals through Ca_V1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca²⁺ channels in nonexcitable cells during development.     

Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na⁺ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the β1 Na⁺ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type β1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the β1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na⁺ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with β1(C121W) subunits. We therefore conclude that Na⁺ channel β1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.     

GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the K_ATP channel blocker tolbutamide, and the L-type Ca²⁺ channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na⁺. The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na⁺-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.     

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory,mechanical, and human disease–derived stimuli

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype Na_V1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that Na_V1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE₂ were substantially reduced in Na_V1.9^−/− mice. Deletion of Na_V1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE₂, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of Na_V1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in Na_V1.9^−/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in Na_V1.9^−/− mice. These results demonstrate that Na_V1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that Na_V1.9 represents a high-value target for development of visceral analgesics.     

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca²⁺ influx via voltage-gated L-type Ca²⁺ channels activates Ca²⁺ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca²⁺]_i transients in hESC-CMs required Ca²⁺ entry through L-type Ca²⁺ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca²⁺ or treatment with diltiazem, an L-type channel inhibitor. Ca²⁺ release from the SR also contributes to the [Ca²⁺]_i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca²⁺ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca²⁺ currents (I_Ca) and corresponding whole-cell [Ca²⁺]_i transients in hESC-CMs and hFVMs, and the amplitude of both I_Ca and the [Ca²⁺]_i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca²⁺]_i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca²⁺ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca²⁺ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca²⁺ release by the I_Ca during EC coupling develops early in human cardiomyocytes.     

Persistent inflammation alters the density and distribution of voltage-activated calcium channels in subpopulations of rat cutaneous DRG neurons

The impact of persistent inflammation on voltage-activated Ca²⁺ channels in cutaneous DRG neurons from adult rats was assessed with whole cell patch clamp techniques, sqRT-PCR and Western blot analysis. Inflammation was induced with a subcutaneous injection of complete Freund’s adjuvant (CFA). DiI was used to identify DRG neurons innervating the site of inflammation. Three days after CFA injection, high threshold Ca²⁺ current (HVA) density was significantly reduced in small and medium, but not large diameter neurons, reflecting a decrease in N-, L- and P/Q-type currents. This decrease in HVA current was associated with an increase in mRNA encoding the α2δ1-subunit complex, but no detectable change in N-type subunit (Ca_V2.2) mRNA. An increase in both α2δ1 and Ca_V2.2 protein was detected in the central nerves arising from L4 and L5 ganglia ipsilateral to the site of inflammation. In current clamp experiments on small and medium diameter cutaneous DRG neurons from naïve rats, blocking ∼40% of HVA current with Cd²⁺ (5 μM), had opposite effects on subpopulations of cutaneous DRG neurons (increasing excitability and action potential duration in some and decreasing excitability in others). The alterations in the density and distribution of voltage-activated Ca²⁺ channels in subpopulations of cutaneous DRG neurons that develop following CFA injection should contribute to changes in sensory transmission observed in the presence of inflammation.     

Investigation of K+ channel expression in human peripheral lymphocytes of healthy donors by means of flow cytometry

Evaluation of different types of K⁺ channel expression was performed in resting and PHA (phytohemagglutinine)-activated human peripheral lymphocytes (HPL) of healthy donors by means of flow cytometry. In resting peripheral lymphocytes, the application of kaliotoxin (a selective blocker for voltage-dependent K+ (K(V)) channels), K(V) resulted in pronounced depolarization of lymphocyte membrane potential, with further promotion in the presence of thapsigargin (compound discharging Ca_i from endoplasmic reticulum). In activated HPL, the expression of various types of K⁺ channels was estimated utilizing cell-cycle analysis data. In contrast to the resting cells, kaliotoxin-induced depolarization of membrane potential in PHA-activated lymphocytes of the G₀/G₁ phase was not enhanced by thapsigargin and in PHA-activated lymphocytes of the S and G2/M phases we were able to observe repolarization of membrane potential after kaliotoxin-induced depolarization of membrane potential. Substitution of kaliotoxin for charybdotoxin (a non-selective drug blocking both K(V) and K(Ca) channels) abrogated the above effects in PHA-activated lymphocytes. Thus, K(V) channels are active in both resting and activated HPLs and K(Ca) channel expression occurs with cell-cycle progress on PHA-induced activation of peripheral lymphocytes.     

Control of somatic membrane potential in nociceptive neurons and its implications for peripheral nociceptive transmission

Peripheral sensory ganglia contain somata of afferent fibres conveying somatosensory inputs to the central nervous system. Growing evidence suggests that the somatic/perisomatic region of sensory neurons can influence peripheral sensory transmission. Control of resting membrane potential (E_rest) is an important mechanism regulating excitability, but surprisingly little is known about how E_rest is regulated in sensory neuron somata or how changes in somatic/perisomatic E_rest affect peripheral sensory transmission. We first evaluated the influence of several major ion channels on E_rest in cultured small-diameter, mostly capsaicin-sensitive (presumed nociceptive) dorsal root ganglion (DRG) neurons. The strongest and most prevalent effect on E_rest was achieved by modulating M channels, K2P and 4-aminopiridine-sensitive K_V channels, while hyperpolarization-activated cyclic nucleotide-gated, voltage-gated Na⁺, and T-type Ca²⁺ channels to a lesser extent also contributed to E_rest. Second, we investigated how varying somatic/perisomatic membrane potential, by manipulating ion channels of sensory neurons within the DRG, affected peripheral nociceptive transmission in vivo. Acute focal application of M or K_ATP channel enhancers or a hyperpolarization-activated cyclic nucleotide-gated channel blocker to L5 DRG in vivo significantly alleviated pain induced by hind paw injection of bradykinin. Finally, we show with computational modelling how somatic/perisomatic hyperpolarization, in concert with the low-pass filtering properties of the t-junction within the DRG, can interfere with action potential propagation. Our study deciphers a complement of ion channels that sets the somatic E_rest of nociceptive neurons and provides strong evidence for a robust filtering role of the somatic and perisomatic compartments of peripheral nociceptive neuron.