1. Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI). Claims that its beneficial psychotropic effects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions. 2. We have used the voltage clamp technique to study the effect of fluoxetine on a neuronal, voltage-dependent potassium (K+) channel (RCK1; Kv1.1), expressed in p6nopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K+ channels containing this subunit are involved in the repolarization process of many types of neurones. 3. Blockade of the K+ currents by fluoxetine was found to be use- and dose-dependent. Wash-out of this compound could not be achieved. Fluoxetine did not affect the ion selectivity of this K+ channel, as the reversal potential was unaltered. 4. Slowing of both activation and deactivation kinetics of the channel by fluoxetine was observed, including tail current crossover upon repolarization. 5. Hodgkin-Huxley type of models and more generalized Markov chain models were used to fit the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of fluoxetine consists of two components: a voltage-independent occurring in the last closed, but available state of the channel, and a voltage-dependent occurring in the open state. 6. This study describes the first biophysical working model for the mechanism of action of fluoxetine on a neuronal, voltage-dependent K+ channel, RCK1. Although this channel is not very potently blocked by fluoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.

The voltage-gated potassium channel, Kv1.1, was recently identified as a causative gene in isolated dominant hypomagnesemia. The channel is situated in the distal convoluted tubule, where it participates in maintaining a favorable electrical gradient for driving magnesium ion into the cell through the transient receptor potential melastatin 6 channel. Pull-down experiments coupled to mass spectrometry using the carboxy-terminal domain of Kv1.1 as bait were used in mouse kidney lysates. Ankyrin-3 (ANK3) was identified as a binding partner of Kv1.1 and was enriched in isolated distal convoluted tubules as compared to whole kidney. Electrophysiology studies performed in HEK293 cells expressing Kv1.1 showed that ANK3 significantly inhibited Kv1.1-mediated currents (267 compared to 125 pA/pF) for control and ANK3, respectively. Finally, to evaluate a potential role of ANK3 in magnesium handling, the intrarenal abundance of ANK3 was measured in mice fed a low-, normal-, or high-magnesium diet for 10 days. Mice maintained on high dietary magnesium significantly doubled their fractional urinary excretion of magnesium, which coincided with a 1.8-fold increase in the renal expression of ANK3 compared to mice on a normal- or low-magnesium diet. Thus, our observations demonstrate a novel role for ANK3 in modulating the biophysical properties of Kv1.1. Such regulation appears to be particularly important in conditions of high dietary magnesium.

Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. The channel is widely expressed in brain but only minimally, if at all, in mouse myocardium. To test whether Kv1.1-potassium deficiency could underlie primary neurogenic cardiac dysfunction, we performed simultaneous video EEG-ECG recordings and found that Kcna1-null mice display potentially malignant interictal cardiac abnormalities, including a fivefold increase in atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, predisposing Kv1.1-deficient mice to sudden unexplained death in epilepsy (SUDEP), which we recorded fortuitously in one animal. To determine whether the interictal AV conduction blocks were of cardiac or neural origin, we examined their response to selective pharmacological blockade of the autonomic nervous system. Simultaneous administration of atropine and propranolol to block parasympathetic and sympathetic branches, respectively, eliminated conduction blocks. When administered separately, only atropine ameliorated AV conduction blocks, indicating that excessive parasympathetic tone contributes to the neurocardiac defect. We found no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart, suggesting a novel site of action leading to Kv1.1-associated cardiac bradyarrhythmias. Together, our data suggest that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for human SUDEP.

Two voltage-dependent potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer Kv1.1-1.2((3)) . Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of Kv1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer Kv1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the Kv1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by Kv1.1-1.2((3)) channels by more than 80 % at 10 μM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6 μM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of Kv1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Nav1.5, and Cav1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries.

Background ADAM22 and ADAM23 are transmembrane proteins that bind the secreted synaptic protein LGI1 and associate with Kv1.1/Kv1.4 potassium channels. However, the roles of these proteins in regulated voltage-gated potassium currents are poorly understood. Methods Cultured cells were transfected to express ADAM22, ADAM23, and Kv1.1/Kv1.4. Voltage-gated potassium currents were measured by whole-cell patch-clamp. Immunostaining Kv1.1 with fluorescent antibodies and fluorescently tagged Kv1.1 subunits was used to measure the effects of ADAM proteins on cell-surface and total expression of Kv1.1 channels. LGI1-conditioned media was added to assess the effect on LGI1 on Kv1.1 currents. Results Cells transfected with Kv1.1/Kv1.4 showed voltage-gated potassium currents (Kv1.1 currents). ADAM23 was a powerful negative regulator of Kv1.1 currents and caused decreased surface expression of Kv1.1 subunits. This decrease in current was not mediated by clathrin-dependent endocytosis. LGI1-conditioned media did not affect the negative regulation of Kv1.1 currents by ADAM23. ADAM22 had no significant effect on Kv1.1 currents by itself, but in the presence of LGI1-conditioned media markedly potentiated Kv1.1 currents without changing channel activation kinetics. Conclusions ADAM22 and ADAM23 have opposite effects on Kv1.1 currents. The relative expression of these proteins, and the availability of LGI1 may shape the expression of Kv1.1 currents in different neuronal membrane domains.

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.

Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients.

The voltage-gated potassium channel Kv1.1, which is abundant in the CNS and peripheral nervous system, controls neuronal excitability and neuromuscular transmission and mediates a number of physiological functions in non-excitable cells. The development of some diseases is accompanied by changes in the expression level and/or activity of the channels in particular types of cells. To meet the requirements of studies related to the expression and localization of the Kv1.1 channels, we report on the subnanomolar affinity of hongotoxin 1 N-terminally labeled with Atto 488 fluorophore (A-HgTx) for the Kv1.1 channel and its applicability for fluorescent imaging of the channel in living cells. Taking into consideration the pharmacological potential of the Kv1.1 channel, a fluorescence-based analytical system was developed for the study of peptide ligands that block the ion conductivity of Kv1.1 and are potentially able to correct abnormal activity of the channel. The system is based on analysis of the competitive binding of the studied compounds and A-HgTx to the mKate2-tagged human Kv1.1 (S369T) channel, expressed in the plasma membrane of Neuro2a cells. The system was validated by measuring the affinities of the known Kv1.1-channel peptide blockers, such as agitoxin 2, kaliotoxin 1, hongotoxin 1, and margatoxin. Peptide pore blocker Ce1, from the venom of the scorpion Centruroides elegans, was shown to possess a nanomolar affinity for the Kv1.1 channel. It is reported that interactions of the Kv1.1 channel with the studied peptide blockers are not affected by the transition of the channel from the closed to open state. The conclusion is made that the structural rearrangements accompanying the channel transition into the open state do not change the conformation of the P-loop (including the selectivity filter) involved in the formation of the binding site of the peptide pore blockers.

Action potential (AP) responses of dentate gyrus granule (DG) cells have to be tightly regulated to maintain hippocampal function. However, which ion channels control the response delay of DG cells is not known. In some neuron types, spike latency is influenced by a dendrotoxin (DTX)-sensitive delay current (ID) mediated by unidentified combinations of voltage-gated K(+) (Kv) channels of the Kv1 family Kv1.1-6. In DG cells, the ID has not been characterized and its molecular basis is unknown. The response phenotype of mature DG cells is usually considered homogenous but intrinsic plasticity likely occurs in particular in conditions of hyperexcitability, for example during temporal lobe epilepsy (TLE). In this study, we examined response delays of DG cells and underlying ion channel molecules by employing a combination of gramicidin-perforated patch-clamp recordings in acute brain slices and single-cell reverse transcriptase quantitative polymerase chain reaction (SC RT-qPCR) experiments. An in vivo mouse model of TLE consisting of intrahippocampal kainate (KA) injection was used to examine epilepsy-related plasticity. Response delays of DG cells were DTX-sensitive and strongly increased in KA-injected hippocampi; Kv1.1 mRNA was elevated 10-fold, and the response delays correlated with Kv1.1 mRNA abundance on the single cell level. Other Kv1 subunits did not show overt changes in mRNA levels. Kv1.1 immunolabeling was enhanced in KA DG cells. The biophysical properties of ID and a delay heterogeneity within the DG cell population was characterized. Using organotypic hippocampal slice cultures (OHCs), where KA incubation also induced ID upregulation, the homeostatic reversibility and neuroprotective potential for DG cells were tested. In summary, the AP timing of DG cells is effectively controlled via scaling of Kv1.1 subunit transcription. With this antiepileptic mechanism, DG cells delay their responses during hyperexcitation.

Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Neural progenitor cells (NPCs) are a source of new neurons and glia in the adult brain. Most NPCs reside in the forebrain subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus, where they contribute to plasticity in the adult brain. To use their potential for repair, it is essential to identify the molecules that regulate their growth, migration and differentiation. Potassium (K+) channels are promising molecule candidates for NPC regulation as they are important components of signal transduction and their diversity is ideal to cover the complex functions required for cell proliferation and differentiation. There is increasing evidence that K+ channels influence cell growth and neurogenesis, however, very little is known regarding K+ channel distribution in NPCs. We therefore explored the expression of a variety of voltage-gated (Kv), inwardly rectifying (Kir) and two-pore (K2P) K+ channels in the SVZ of adult mice and in neurosphere cultures of NPCs during growth and differentiation. Immunocytochemical analysis revealed a differential expression pattern of K+ channels in nestin+ SVZ precursor cells, early SVZ doublecortin+ neurons and (sub)ependymal cells. These findings were confirmed in neurosphere cultures at the protein and mRNA levels. The expression of some K+ channel proteins, such as Kir4.1, Kir6.1, TREK1 or TASK1, suggests a role of K+ channels in the complex regulation of NPC proliferation, maturation and differentiation.

Oncogene-induced senescence (OIS) constitutes a failsafe program that restricts tumor development. However, the mechanisms that link oncogenesis to senescence are not completely understood. We carried out a loss-of-function genetic screen that identified the potassium channel KCNA1 as a determinant of OIS escape that can license tumor growth. Oncogenic stress triggers an increase in KCNA1 expression and its relocation from the cytoplasm to the membrane. Mechanistically, this relocation is due to a loss of protein kinase A (PKA)-induced phosphorylation at residue S446 of KCNA1. Accordingly, sustaining PKA activity or expressing a KCNA1 phosphomimetic mutant maintained KCNA1 in the cytoplasm and caused escape from OIS. KCNA1 relocation to the membrane induced a change in membrane potential that invariably resulted in cellular senescence. Restoring KCNA1 expression in transformation-competent cells triggered variation in membrane potential and blocked RAS-induced transformation, and PKA activation suppressed both effects. Furthermore, KCNA1 expression was reduced in human cancers, and this decrease correlated with an increase in breast cancer aggressiveness. Taken together, our results identify a novel pathway that restricts oncogenesis through a potassium channel-dependent senescence pathway.

The KCNA1 gene encodes the α subunit of the voltage-gated Kv1.1 potassium channel that critically regulates neuronal excitability in the central and peripheral nervous systems. Mutations in KCNA1 have been classically associated with episodic ataxia type 1 (EA1), a movement disorder triggered by physical and emotional stress. Additional features variably reported in recent years include epilepsy, myokymia, migraine, paroxysmal dyskinesia, hyperthermia, hypomagnesemia, and cataplexy. Interestingly, a few individuals with neuromyotonia, either isolated or associated with skeletal deformities, have been reported carrying variants in the S2-S3 transmembrane segments of Kv1.1 channels in the absence of any other symptoms. Here, we have identified by whole-exome sequencing a novel de novo variant, T268K, in KCNA1 in a boy displaying recurrent episodes of neuromyotonia, muscle hypertrophy, and skeletal deformities. Through functional analysis in heterologous cells and structural modeling, we show that the mutation, located at the extracellular end of the S3 helix, causes deleterious effects, disrupting Kv1.1 function by altering the voltage dependence of activation and kinetics of deactivation, likely due to abnormal interactions with the voltage sensor in the S4 segment. Our study supports previous evidence suggesting that specific residues within the S2 and S3 segments of Kv1.1 result in a distinctive phenotype with predominant musculoskeletal presentation.

Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase-independent (sGC-independent) effects are stereoselective - that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) - and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Whereas Kvbeta2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kvbeta2 subunits to native potassium channel function. Using antisense approaches and in situ recordings from Xenopus embryo spinal cord neurons, we tested the in vivo roles of Kvbeta2 subunits in modulation of voltage-dependent potassium current (IKv). We focused on 1) two different populations of dorsal spinal neurons that express both Kvbeta2 and Kv1 alpha-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvbeta2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvbeta2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 alpha-subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvbeta2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.

Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury.