Selective Loss of Presynaptic Potassium Channel Clusters at the Cerebellar Basket Cell Terminal Pinceau in Adam11 Mutants Reveals Their Role in Ephaptic Control of Purkinje Cell Firing.

A specialized axonal ending, the basket cell "pinceau," encapsulates the Purkinje cell axon initial segment (AIS), exerting final inhibitory control over the integrated outflow of the cerebellar cortex. This nonconventional axo-axonic contact extends beyond the perisomatic chemical GABAergic synaptic boutons to the distal AIS, lacks both sodium channels and local exocytotic machinery, and yet contains a dense cluster of voltage-gated potassium channels whose functional contribution is unknown. Here, we show that ADAM11, a transmembrane noncatalytic disintegrin, is the first reported Kv1-interacting protein essential for localizing Kv1.1 and Kv1.2 subunit complexes to the distal terminal. Selective absence of these channels at the pinceau due to mutation of ADAM11 spares spontaneous GABA release from basket cells at the perisomatic synapse yet eliminates ultrarapid ephaptic inhibitory synchronization of Purkinje cell firing. Our findings identify a critical role for presynaptic K(+) channels at the pinceau in ephaptic control over the speed and stability of spike rate coding at the Purkinje cell AIS in mice. SIGNIFICANCE STATEMENT: This study identifies ADAM11 as the first essential molecule for the proper localization of potassium ion channels at presynaptic nerve terminals, where they modulate excitability and the release of neural transmitters. Genetic truncation of the transmembrane disintegrin and metalloproteinase protein ADAM11 resulted in the absence of Kv1 channels that are normally densely clustered at the terminals of basket cell axons in the cerebellar cortex. These specialized terminals are responsible for the release of the neurotransmitter GABA onto Purkinje cells and also display electrical signaling. In the ADAM11 mutant, GABAergic release was not altered, but the ultrarapid electrical signal was absent, demonstrating that the dense presynaptic cluster of Kv1 ion channels at these terminals mediate electrical transmission. Therefore, ADAM11 plays a critical role at this central synapse.

High-resolution molecular genomic autopsy reveals complex sudden unexpected death in epilepsy risk profile.

Advanced variant detection in genes underlying risk of sudden unexpected death in epilepsy (SUDEP) can uncover extensive epistatic complexity and improve diagnostic accuracy of epilepsy-related mortality. However, the sensitivity and clinical utility of diagnostic panels based solely on established cardiac arrhythmia genes in the molecular autopsy of SUDEP is unknown. We applied the established clinical diagnostic panels, followed by sequencing and a high density copy number variant (CNV) detection array of an additional 253 related ion channel subunit genes to analyze the overall genomic variation in a SUDEP of the 3-year-old proband with severe myoclonic epilepsy of infancy (SMEI). We uncovered complex combinations of single nucleotide polymorphisms and CNVs in genes expressed in both neurocardiac and respiratory control pathways, including SCN1A, KCNA1, RYR3, and HTR2C. Our findings demonstrate the importance of comprehensive high-resolution variant analysis in the assessment of personally relevant SUDEP risk. In this case, the combination of de novo single nucleotide polymorphisms (SNPs) and CNVs in the SCN1A and KCNA1 genes, respectively, is suspected to be the principal risk factor for both epilepsy and premature death. However, consideration of the overall biologically relevant variant complexity with its extensive functional epistatic interactions reveals potential personal risk more accurately.

Knockout of Zn transporters Zip-1 and Zip-3 attenuates seizure-induced CA1 neurodegeneration.

CA1 pyramidal neurons are the final integrators of information flow leaving the hippocampus, yet are singularly vulnerable to activity-dependent cell death. Zinc (Zn) entry into cells may add to this vulnerability. Here, we find that Slc39a1 and Slc39a3, members of the Zip (Zrt/Irt-like protein) plasmalemmal Zn transporter family, are predominantly expressed in the hippocampus. We examined Zip-1,3-deficient mice to investigate their role in neurodegeneration following intense synaptic activation. When isolated by blockade of NMDA receptors and voltage-gated calcium channels, the absence of both transporters slowed passive Zn uptake into CA1 neurons measured with intracellular fluorescent Zn dyes. In vivo CA1 cell damage following kainic acid exposure was greatly attenuated. Consistent with the hypothesis that Zn entry contributes to neurodegeneration, Znt-3-deficient mice lacking synaptic Zn also show less hippocampal cell damage following kainic acid injection. Zip transporters may provide selective therapeutic targets to protect these neurons from early Zn-induced neurodegeneration following injury.

Kv1.1 potassium channel deficiency reveals brain-driven cardiac dysfunction as a candidate mechanism for sudden unexplained death in epilepsy.

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.

Key role of Kv1 channels in vasoregulation.

Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K+ (K(DR)) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow.

Novel brain expression of ClC-1 chloride channels and enrichment of CLCN1 variants in epilepsy.

OBJECTIVE: To explore the potential contribution of genetic variation in voltage-gated chloride channels to epilepsy, we analyzed CLCN family (CLCN1-7) gene variant profiles in individuals with complex idiopathic epilepsy syndromes and determined the expression of these channels in human and murine brain. METHODS: We used parallel exomic sequencing of 237 ion channel subunit genes to screen individuals with a clinical diagnosis of idiopathic epilepsy and evaluate the distribution of missense variants in CLCN genes in cases and controls. We examined regional expression of CLCN1 in human and mouse brain using reverse transcriptase PCR, in situ hybridization, and Western immunoblotting. RESULTS: We found that in 152 individuals with sporadic epilepsy of unknown origin, 96.7% had at least one missense variant in the CLCN genes compared with 28.2% of 139 controls. Nonsynonymous single nucleotide polymorphisms in the "skeletal" chloride channel gene CLCN1 and in CLCN2, a putative human epilepsy gene, were detected in threefold excess in cases relative to controls. Among these, we report a novel de novo CLCN1 truncation mutation in a patient with pharmacoresistant generalized seizures and a dystonic writer's cramp without evidence of variants in other channel genes linked to epilepsy. Molecular localization revealed the unexpectedly widespread presence of CLCN1 mRNA transcripts and the ClC-1 subunit protein in human and murine brain, previously believed absent in neurons. CONCLUSIONS: Our findings support a possible comorbid contribution of the "skeletal" chloride channel ClC-1 to the regulation of brain excitability and the need for further elucidation of the roles of CLCN genes in neuronal network excitability disorders.

Myogenic regulation of arterial diameter: role of potassium channels with a focus on delayed rectifier potassium current.

The phenomenon of myogenic constriction of arterial resistance vessels in response to increased intraluminal pressure has been known for over 100 years, yet our understanding of the molecular mechanisms involved remains incomplete. The focus of this paper concerns the potassium (K+) channels that provide a negative feedback control of the myogenic depolarization of vascular smooth muscle cells that is provoked by elevations in intraluminal pressure, and specifically, the contribution of delayed rectifier (KDR) channels. Our knowledge of the important role played by KDR channels, as well as their molecular identity and acute modulation via changes in gating, has increased dramatically in recent years. Several lines of evidence point to a crucial contribution by heteromultimeric KV1 subunit-containing KDR channels in the control of arterial diameter and myogenic reactivity, but other members of the KV superfamily are also expressed by vascular myocytes, and less is known concerning their specific functions. The effect of pharmacological modulation of KDR channels is discussed, with particular reference to the actions of anorexinogens on KV1- and KV2-containing KDR channels. Finally, the need for a greater understanding of the mechanisms that control KDR channel gene expression is stressed in light of evidence indicating that there is a reduced expression of KDR channels in diseases associated with abnormal myogenic reactivity and vascular remodelling.