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A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

ABSTRACT: Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an ?25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.

SUBMITTER: Tyagi S

PROVIDER: S-EPMC6571999 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

Tyagi Sidharth S Bendrick Tyler R TR Filipova Dilyana D Papadopoulos Symeon S Bannister Roger A RA

The Journal of general physiology 20190423 6

Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutat ...[more]

PMID: 31015257

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Project description:P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 ?1 subunit and the auxiliary ?2?-2 and ?4 subunits. ?4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat ?4b cDNA. Heterologously expressed wild-type ?4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the ?4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, ?4b and ?4b-L125P augmented the calcium current amplitudes, however, ?4b-L125P failed to stably complex with ?1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of ?4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype ?4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of ?4b in cultured myotubes and hippocampal neurons. While binding of ?4b to the known interaction partner PPP2R5D (B56?) was not affected by the mutation, complex formation between ?4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of ?4b.

Project description:ObjectiveN-methyl-d-aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1-3b[I481V]), p.Ala666Ser (GluN1-3b[A666S]), and p.Tyr668His (GluN1-3b[Y668H]).MethodsWild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy.ResultsOur data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single-receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors.SignificanceOur study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder.

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A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

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A mutation in Ca<sub>V</sub>2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

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