A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression‐burst enhances Kv7/M channel activity

Mutations in the KCNQ2 gene encoding the voltage‐gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2^V175L) found in a patient presenting EOEE. We observed that the mutation produced a 25–40 mV hyperpolarizing shift of the conductance–voltage relationship of both the homomeric Kv7.2^V175L and heteromeric Kv7.2^V175L/Kv7.3 channels compared to wild‐type channels and a 10 mV hyperpolarizing shift of Kv7.2^V175L/Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain‐of‐function variants.     

Cell type–specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors

The Kv2.1 voltage‐gated K⁺ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity‐dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca²⁺‐release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy–immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR‐mediated Ca²⁺ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca²⁺/calcineurin‐dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell‐ and circuit‐specific mechanism for coupling intracellular Ca²⁺ release to phosphorylation‐dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. J. Comp. Neurol. 522:3555–3574, 2014. © 2014 Wiley Periodicals, Inc.     

Effects of Acetazolamide on Transient K+ Currents and Action Potentials in Nodose Ganglion Neurons of Adult Rats

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K⁺ current (I_A) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4‐AP attenuated or blocked the CO₂‐induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch‐clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT‐PCR of mRNA and the expression of voltage‐gated K⁺ (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I_A by approximately 27% and the additional application of 4‐AP (1 mM) further inhibited I_A by 48%. The application of 0.1 μM α‐dendrotoxin (α‐DTX), a slow inactivating transient K⁺ current (I_D) blocker, inhibited the baseline I_A by approximately 27%, and the additional application of 1 mM AZ further decreased the I_A by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage‐gated K⁺ channel proteins were present, and most (80–90%) of the four Kv channels immunoreactive neurons showed the co‐expression of carbonic anhydrase‐II (CA‐II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I_A via the inhibition of four voltage‐gated K⁺ channel (Kv) proteins without affecting I_D.     

Kv4.2 block of long-term potentiation is partially dependent on synaptic NMDA receptor remodeling

Proper expression of synaptic NMDA receptors (NMDARs) is necessary to regulate synaptic Ca²⁺ influx and the induction the long-term potentiation (LTP) in the mammalian hippocampus. Previously we reported that expressing the A-type K⁺ channel subunit Kv4.2 in CA1 neurons of organotypic slice cultures reduced synaptic NR2B-containing NMDAR expression and completely blocked LTP induced by a pairing protocol. As pretreatment with an NMDAR antagonist (APV) overnight blocked the reduction of NR2B-containing receptors in neurons expressing EGFP-labeled Kv4.2 (Kv4.2g), we hypothesized that LTP would be rescued in Kv4.2g neurons by overnight treatment with APV. We report here that the overnight APV pretreatment in Kv4.2g-expressing neurons only partially restored potentiation. This partial potentiation was completely blocked by inhibition of the CAMKII kinase. These results indicate that A-type K⁺ channels must regulate synaptic integration and plasticity through another mechanism in addition to their regulation of synaptic NR2 subunit composition. We suggest that dendritic excitability, which is regulated by Kv4.2 expression, also contributes to synaptic plasticity.     

GABA and glutamate receptors have different effects on excitability and are differentially regulated by calcium in spider mechanosensory neurons

GABA and glutamate receptors belonging to the ligand‐gated chloride‐channel family are primary targets of insecticides and antiparasitics, so their molecular structure, pharmacology and biophysical properties have attracted significant attention. However, little is known about the physiological roles of these channels or how they regulate neuronal excitability and animal behavior. Mechanosensory neurons of VS‐3 slit sensilla in the patella of the tropical wandering spider, Cupiennius salei, react to the GABA_A‐receptor agonists, GABA and muscimol, with depolarization and an increase in intracellular [Ca²⁺] and, during random noise stimulation, with a mixed inhibitory–excitatory response. We established that the GABA_A‐receptors in all VS‐3 neurons are identical, but there are at least two types of glutamate receptors and some neurons do not respond to glutamate at all. Immunohistochemistry with antibodies against Drosophila inhibitory glutamate receptor (GluCls) α‐subunit suggests that in addition to VS‐3 neurons, these receptors may also be present in the efferent neurons surrounding the sensory neurons. Most VS‐3 neurons were inhibited but not depolarized by glutamate during random stimulation, but some depolarized and had a similar excitatory–inhibitory response to glutamate as to muscimol. The membrane‐permeable Ca²⁺‐chelator BAPTA‐AM abolished muscimol effects but potentiated glutamate effects, indicating that GABA and glutamate receptors are differentially modulated by Ca²⁺, leading to diverse regulation of neuronal excitability. We hypothesize that this could be achieved by different Ca²⁺‐triggered phosphorylation processes at each receptor type. These findings are important for understanding the significance of Ca²⁺‐mediated regulation of transmitter receptor molecules and its role in controlling excitability.     

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Colonic inflammation up-regulates voltage-gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors

Background Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na⁺ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na⁺ channels expressed in bladder sensory neurons. Methods Sprague–Dawley rats were treated with intracolonic saline (control), resiniferatoxin (RTX, 10^?7 mol L^?1), TNBS (colonic irritant) or double treatment (RTX followed by TNBS). Key Results TNBS‐induced colitis increased the amplitude of total Na⁺ current by two‐fold and of tetrodotoxin resistant (TTX‐R) Na⁺ current by 78% (P ≤ 0.05 to control) in lumbosacral bladder neurons during acute phase (3 days post‐TNBS). Instillation of RTX in the distal colon caused an enhancement in the amplitude of total Na⁺ current at ?20 mV from ?112.1 ± 18.7 pA/pF (control) to ?183.6 ± 27.8 pA/pF (3 days post‐RTX, P ≤ 0.05) without changes in TTX resistant component. The amplitude of net Na⁺ current was also increased by 119% at day 3 in the group with double treatment (RTX followed by TNBS, P ≤ 0.05 to control) which was significantly higher than in either group with a single treatment. Conclusions & Inferences These results provide evidence that colonic inflammation activates TRPV1 receptors at the peripheral sensory terminals leading to an up‐regulation of voltage gated Na⁺ channels on the cell soma of bladder sensory neurons. This mechanism may underlie the occurrence of peripheral cross‐sensitization in the pelvis and functional chronic pelvic pain.     

KChIP4a regulates Kv4.2 channel trafficking through PKA phosphorylation

Voltage-gated potassium (Kv) channels play important roles in regulating the excitability of myocytes and neurons. Kv4.2 is the primary α-subunit of the channel that produces the A-type K⁺ current in CA1 pyramidal neurons of the hippocampus, which is critically involved in the regulation of dendritic excitability and plasticity. K⁺ channel-interacting proteins, KChIPs (KChIP1–4), associate with the N-terminal of Kv4.2 and modulate the channel's biophysical properties, turnover rate and surface expression. In the present study, we investigated the role of Kv4.2 C-terminal PKA phosphorylation site S552 in the KChIP4a-mediated effects on Kv4.2 channel trafficking. We found that while interaction between Kv4.2 and KChIP4a does not require PKA phosphorylation of Kv4.2^S552, phosphorylation of this site is necessary for both enhanced stabilization and membrane expression of Kv4.2 channel complexes produced by KChIP4a. Enhanced surface expression and protein stability conferred by co-expression of Kv4.2 with other KChIP isoforms did not require PKA phosphorylation of Kv4.2 S552. Finally, we identify A-kinase anchoring proteins (AKAPs) as Kv4.2 binding partners, allowing for discrete local PKA signaling. These data demonstrate that PKA phosphorylation of Kv4.2 plays an important role in the trafficking of Kv4.2 through its specific interaction with KChIP4a.     

Localization of nectin‐2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage‐gated A‐type K+ channel Kv4.2 in the medial habenula

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin‐like cell adhesion molecules (CAMs) nectin‐2α and nectin‐2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin‐2α, but not nectin‐2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage‐gated A‐type K⁺ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin‐2α at this boundary was not colocalized with the nectin‐2α‐binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin‐2α forms a cell adhesion apparatus different from the Kv4.2‐associated MSs. Genetic ablation of nectin‐2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin‐2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.     

Neurokinins inhibit low threshold inactivating K currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K⁺ currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK₂ agonist, [βAla⁸]-neurokinin A (4–10) also inhibited low threshold inactivating K⁺ currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K⁺ currents in CAPS-responsive phasic neurons was fully reversed by an NK₂ receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK₁ selective agonist ([Sar⁹, Met¹¹]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K⁺ currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK₂ receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.     

Comparing axonal excitability in past polio to amyotrophic lateral sclerosis

Introduction: Poliomyelitis causes selective destruction of anterior horn cells and usually has a stable disease course post‐infection. We assessed the excitability characteristics in patients with a stable course after past poliomyelitis and compared them with changes described in amyotrophic lateral sclerosis (ALS). Methods: The excitability characteristics of motor and sensory nerves were studied in 10 subjects with stable past poliomyelitis. Results: Motor rheobase was increased, but there were no significant changes in strength–duration properties or depolarizing threshold electrotonus, as have been seen in previous studies of ALS. Conclusions: There is minimal change in axonal excitability properties in patients with stable past poliomyelitis. The results may signify sufficient compensation in the stable state of the disease. Increased subexcitability in 1 subject with demonstrable hyperexcitability may represent compensation for increased ectopic activity rather than a different process in surviving motor neurons. Muscle Nerve 50: 602–604, 2014     

Release of endogenous opioids during a chronic IBD model suppresses the excitability of colonic DRG neurons

Background Endogenous opioids are implicated in pain‐regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage‐gated K⁺ currents was involved. Methods The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF). Key Results In current‐clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. β‐endorphin increased 14‐fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the μ‐opioid receptor agonist D‐Ala², N‐ MePhe⁴, Gly‐ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the μ‐opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I_A K⁺ currents. Conclusions & Inferences The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.     

Potential role of KCNQ/M-channels in regulating neuronal differentiation in mouse hippocampal and embryonic stem cell-derived neuronal cultures

Voltage-gated K⁺ channels are key regulators of neuronal excitability, playing major roles in setting resting membrane potential, repolarizing the cell membrane after action potentials and affecting transmitter release. The M-type channel or M-channel is a unique voltage- and ligand-regulated K⁺ channel. It is composed of the molecular counterparts KCNQ2 and KCNQ3 (also named Kv7.2 and Kv7.3) channels and expressed in the soma and dendrites of neurons. The present investigation examined the hypothesis that KCNQ2/3 channels played a regulatory role in neuronal differentiation and maturation. In cultured mouse embryonic stem (ES) cells undergoing neuronal differentiation and primary embryonic (E15–17) hippocampal cultures, KCNQ2 and KCNQ3 channels and underlying M-currents were identified. Blocking of KCNQ channels in these cells for 5 days using the specific channel blocker XE991 (10 μM) or linopirdine (30 μM) significantly decreased synaptophysin and syntaxin expression without affecting cell viability. Chronic KCNQ2/3 channel block reduced the expression of vesicular GABA transporter (v-GAT), but not vesicular glutamate transporter (v-GluT). Enhanced ERK1/2 phosphorylation was observed in XE991- and linopirdine-treated neural progenitor cells. In electrophysiological recordings, cells undergoing chronic block of KCNQ2/3 channels showed normal amplitude of mPSCs while the frequency of mPSCs was reduced. On the other hand, KCNQ channel opener N-Ethylmaleimide (NEM, 2 μM) increased mPSC frequency. Fluorescent imaging using fluorescent styryl-dye FM4-64 revealed that chronic blockade of KCNQ2/3 channels decreased endocytosis but facilitated exocytosis. These data indicate that KCNQ2/3 channels participate in the regulation of neuronal differentiation and show a tonic regulation on pre-synaptic transmitter release and recycling in developing neuronal cells.     

Arachidonic acid potently inhibits both postsynaptic-type Kv4.2 and presynaptic-type Kv1.4 IA potassium channels

Arachidonic acid (AA) is a free fatty acid membrane‐permeable second messenger that is liberated from cell membranes via receptor‐ and Ca²⁺‐dependent events. We have shown previously that extremely low [AA]_i (1 pm ) inhibits the postsynaptic voltage‐gated K⁺ current (I_A) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic I_A is mediated by Kv4.2 gene products, whereas presynaptic I_A is mediated by Kv1.4 channel subunits. To address the interaction of AA with these α‐subunits we studied the modulation of A‐currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole‐cell voltage‐clamp recording. For both currents 1 pm [AA]_i inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by ?9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co‐application of Trolox C (10 μm ), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4‐mediated I_A by ?32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on I_A amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4‐mediated I_A. Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic I_A in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel α‐subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity.     

Estrogen‐related receptor β deletion modulates whole‐body energy balance via estrogen‐related receptor γ and attenuates neuropeptide Y gene expression

Estrogen‐related receptors (ERRs) α, β and γ are orphan nuclear hormone receptors with no known ligands. Little is known concerning the role of ERRβ in energy homeostasis, as complete ERRβ‐null mice die mid‐gestation. We generated two viable conditional ERRβ‐null mouse models to address its metabolic function. Whole‐body deletion of ERRβ in Sox2‐Cre:ERRβ^lox/lox mice resulted in major alterations in body composition, metabolic rate, meal patterns and voluntary physical activity levels. Nestin‐Cre:ERRβ^lox/lox mice exhibited decreased expression of ERRβ in hindbrain neurons, the predominant site of expression, decreased neuropeptide Y (NPY) gene expression in the hindbrain, increased lean body mass, insulin sensitivity, increased energy expenditure, decreased satiety and decreased time between meals. In the absence of ERRβ, increased ERRγ signaling decreased satiety and the duration of time between meals, similar to meal patterns observed for both the Sox2‐Cre:ERRβ^lox/lox and Nestin‐Cre:ERRβ^lox/lox strains of mice. Central and/or peripheral ERRγ signaling may modulate these phenotypes by decreasing NPY gene expression. Overall, the relative expression ratio between ERRβ and ERRγ may be important in modulating ingestive behavior, specifically satiety, gene expression, as well as whole‐body energy balance.     

Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons

Elimination of the Kv1.3 voltage‐dependent potassium channel gene produces striking changes in the function of the olfactory bulb, raising the possibility that this channel also influences other sensory systems. We have examined the cellular and subcellular localization of Kv1.3 in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, a nucleus in which neurons fire at high rates with high temporal precision. A clear gradient of Kv1.3 immunostaining along the lateral to medial tonotopic axis of the MNTB was detected. Highest levels were found in the lateral region of the MNTB, which corresponds to neurons that respond selectively to low‐frequency auditory stimuli. Previous studies have demonstrated that MNTB neurons and their afferent inputs from the cochlear nucleus express three other members of the Kv1 family, Kv1.1, Kv1.2, and Kv1.6. Nevertheless, confocal microscopy of MNTB sections coimmunostained for Kv1.3 with these subunits revealed that the distribution of Kv1.3 differed significantly from other Kv1 family subunits. In particular, no axonal staining of Kv1.3 was detected, and most prominent labeling was in structures surrounding the somata of the principal neurons, suggesting specific localization to the large calyx of Held presynaptic endings that envelop the principal cells. The presence of Kv1.3 in presynaptic terminals was confirmed by coimmunolocalization with the synaptic markers synaptophysin, syntaxin, and synaptotagmin and by immunogold electron microscopy. Kv1.3 immunogold particles in the terminals were arrayed along the plasma membrane and on internal vesicular structures. To confirm these patterns of staining, we carried out immunolabeling on sections from Kv1.3^−/− mice. No immunoreactivity could be detected in Kv1.3^−/− mice either at the light level or in immunogold experiments. The finding of a tonotopic gradient in presynaptic terminals suggests that Kv1.3 may regulate neurotransmitter release differentially in neurons that respond to different frequencies of sound. J. Comp. Neurol. 518:3205–3220, 2010. © 2010 Wiley‐Liss, Inc.     

Infantile spasms and encephalopathy without preceding neonatal seizures caused by KCNQ2 R198Q,a gain‐of‐function variant

Variants in KCNQ2 encoding for K_v7.2 neuronal K⁺ channel subunits lead to a spectrum of neonatal‐onset epilepsies, ranging from self‐limiting forms to severe epileptic encephalopathy. Most KCNQ2 pathogenic variants cause loss‐of‐function, whereas few increase channel activity (gain‐of‐function). We herein provide evidence for a new phenotypic and functional profile in KCNQ2‐related epilepsy: infantile spasms without prior neonatal seizures associated with a gain‐of‐function gene variant. With use of an international registry, we identified four unrelated patients with the same de novo heterozygous KCNQ2 c.593G>A, p.Arg198Gln (R198Q) variant. All were born at term and discharged home without seizures or concern of encephalopathy, but developed infantile spasms with hypsarrhythmia (or modified hypsarrhythmia) between the ages of 4 and 6 months. At last follow‐up (ages 3–11 years), all patients were seizure‐free and had severe developmental delay. In vitro experiments showed that Kv7.2 R198Q subunits shifted current activation gating to hyperpolarized potentials, indicative of gain‐of‐function; in neurons, K_v7.2 and K_v7.2 R198Q subunits similarly populated the axon initial segment, suggesting that gating changes rather than altered subcellular distribution contribute to disease molecular pathogenesis. We conclude that KCNQ2 R198Q is a model for a new subclass of KCNQ2 variants causing infantile spasms and encephalopathy, without preceding neonatal seizures. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here     

Regulation of T-type Ca<Superscript>2+</Superscript> channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca²⁺ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca²⁺ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca²⁺ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca²⁺ current densities. Increased T-type Ca²⁺ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca²⁺ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca²⁺ channels is mediated by several factors, including decreased expression of Ca_v3.2 T-type Ca²⁺ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca²⁺ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca²⁺ channels, which can results in morphological and functional changes in electrical excitability.     

Voltage-gated K channels in chemoreceptor sensory neurons of rat petrosal ganglion

A subpopulation of sensory neurons in the petrosal ganglion transmits information between peripheral chemoreceptors (glomus cells) in the carotid body and relay neurons in the nucleus of the solitary tract. Expression of voltage-gated K⁺ channels in these neurons was characterized by immunohistochemical localization. Five members of the Kv1 family, Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 and members of two other families, Kv2.1 and Kv4.3, were identified in over 90% of the chemoreceptor neurons. Although the presence of these channel proteins was consistent throughout the population, individual neurons showed considerable variation in K⁺ current profiles.