Pathogenic Cav3.2 channel mutation in a child with primary generalized epilepsy.
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ABSTRACT:
SUBMITTER: Souza IA
PROVIDER: S-EPMC6814130 | biostudies-literature | 2019 Oct
REPOSITORIES: biostudies-literature
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Project description:This study describes the functional interaction between Cav3.2 calcium channels and the Epithelial Sodium Channel (ENaC). ?-ENaC subunits showed overlapping expression with endogenous Cav3.2 calcium channels in the thalamus and hypothalamus as detected by immunostaining. Moreover, ?- and ?-ENaC subunits could be co-immunoprecipitated with Cav3.2 calcium channels from brain lysates, dorsal horn and lumbar dorsal root ganglia. Mutation of a cluster of lysines present in the intracellular N-terminus region of ?-ENaC (K4R/ K5R/ K9R/ K16R/ K23R) reduced interactions with Cav3.2 calcium channels. ???-ENaC channels enhanced Cav3.2 calcium channel trafficking to the plasma membrane in tsA-201 cells. This effect was reciprocal such that Cav3.2 channel expression also enhanced ?-ENaC trafficking to the cell surface. T-type current density was increased when fully assembled ???-ENaC channels were transiently expressed in CAD cells, a neuronal derived cell line. Altogether, these findings reveal ENaC as an interactor and potential regulator of Cav3.2 calcium channels expressed in neuronal tissues.
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Project description:Cav3.2 calcium channels play a key role in nociceptive signaling in the primary afferent pain pathway. We have previously reported the regulation of Cav3.2 calcium channels by the deubiquitinase USP5 and its importance for regulating peripheral transmission of pain signals. Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation. We show that endogenous USP5 protein expressed in dorsal root ganglia undergoes SUMOylation, and the level of USP5 SUMOylation is reduced following peripheral nerve injury. SUMO prediction software identified several putative lysines that have the propensity to be targets for SUMO conjugation. A series of single lysine substitutions in an mCherry tagged USP5 construct followed by expression in tsA-201 cells identified lysine K113 as a key target for USP5 SUMO2/3 modification. Finally, Cav3.2 calcium channel immunoprecipitates revealed a stronger interaction of Cav3.2 with a SUMO2/3 resistant USP5-K113R mutant, indicating that SUMO2/3 modification of USP5 reduces its affinity for the calcium channel Cav3.2. Collectively, our data suggest that dysregulation of USP5 SUMOylation after peripheral nerve injury may contribute to the well described alteration in Cav3.2 channel activity during neuropathic pain states.
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Project description:Variants in RORB have been reported in eight individuals with epilepsy, with phenotypes ranging from eyelid myoclonia with absence epilepsy to developmental and epileptic encephalopathies. We identified novel RORB variants in 11 affected individuals from four families. One was from whole genome sequencing and three were from RORB screening of three epilepsy cohorts: developmental and epileptic encephalopathies (n = 1021), overlap of generalized and occipital epilepsy (n = 84), and photosensitivity (n = 123). Following interviews and review of medical records, individuals' seizure and epilepsy syndromes were classified. Three novel missense variants and one exon 3 deletion were predicted to be pathogenic by in silico tools, not found in population databases, and located in key evolutionary conserved domains. Median age at seizure onset was 3.5 years (0.5-10 years). Generalized, predominantly absence and myoclonic, and occipital seizures were seen in all families, often within the same individual (6/11). All individuals with epilepsy were photosensitive, and seven of 11 had cognitive abnormalities. Electroencephalograms showed generalized spike and wave and/or polyspike and wave. Here we show a striking RORB phenotype of overlap of photosensitive generalized and occipital epilepsy in both individuals and families. This is the first report of a gene associated with this overlap of epilepsy syndromes.
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Project description:Concerted depolarization and Ca(2+) rise during neuronal action potentials activate large-conductance Ca(2+)- and voltage-dependent K(+) (BK) channels, whose robust K(+) currents increase the rate of action potential repolarization. Gain-of-function BK channels in mouse knockout of the inhibitory beta 4 subunit and in a human mutation (alpha(D434G)) have been linked to epilepsy. Here, we investigate mechanisms underlying the gain-of-function effects of the equivalent mouse mutation (alpha(D369G)), its modulation by the beta 4 subunit, and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and Ca(2+)-dependent gating largely account for the gain-of-function effects. D369G causes a greater than twofold increase in the closed-to-open equilibrium constant (6.6e(-7)-->1.65e(-6)) and an approximate twofold decrease in Ca(2+)-dissociation constants (closed channel: 11.3-->5.2 microM; open channel: 0.92-->0.54 microM). The beta 4 subunit inhibits mutant channels through a slowing of activation kinetics. In physiological recording solutions, we established the Ca(2+) dependence of current recruitment during action potential-shaped stimuli. D369G and beta 4 have opposing effects on BK current recruitment, where D369G reduces and beta 4 increases K(1/2) (K(1/2) microM: alpha(WT) 13.7, alpha(D369G) 6.3, alpha(WT)/beta 4 24.8, and alpha(D369G)/beta 4 15.0). Collectively, our results suggest that the D369G enhancement of intrinsic gating and Ca(2+) binding underlies greater contributions of BK current in the sharpening of action potentials for both alpha and alpha/beta 4 channels.
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