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A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of CaV2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca2+ Influx.

Bahamonde MI, Serra SA, Drechsel O, Rahman R, Marcé-Grau A, Prieto M, Ossowski S, Macaya A, Fernández-Fernández JM - PLoS ONE (2015)

Bottom Line: ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels.ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance.Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum.

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De novo heterozygous CACNA1A deletion in congenital ataxia with cerebellar atrophy.(A) Pedigree of the affected individual carrying the de novo heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).
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pone.0146035.g002: De novo heterozygous CACNA1A deletion in congenital ataxia with cerebellar atrophy.(A) Pedigree of the affected individual carrying the de novo heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).

Mentions: Due to the large number of genes potentially causing cerebellar ataxia, we performed whole exome sequencing (WES) of a parent-child trio with an unknown cause of congenital ataxia in the child. Variant detection on the sequencing data using GATK [45] and ClinDel [47] yielded 115,818 and 12,753 variants, respectively. Variants affecting the protein sequence (nonsynonymous or splicing) were filtered based on possible inheritance modes, including de novo, recessive, compound heterozygous or X-linked. Variants coherent with one of the inheritance modes were further prioritized based on population allele frequency in EVS and 1000GP (i.e. rare variants with < 1% AF), damage potential based on PolyPhen-2, SIFT, Condel and CADD [49–52], evolutionary conservation based on phastCons [53] and finally sequence complexity and repetitiveness (e.g. variants in segmental duplications are removed), finally yielding a total of seven de novo, eight recessive and 15 compound heterozygous variants that are rare or novel and highly damaging. The affected 30 genes were subsequently subjected to additional prioritization using ENDEAVOUR [54], which has been trained on genes extracted from various disease gene resources [55,56]. The ENDEAVOUR prioritization highlighted a de novo triplet deletion in CACNA1A (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) as most likely candidate, as being part of the list of training genes. Furthermore, no other genes after filtering could be linked to the investigated phenotype. Manual review of the respective alignment data supports the presence of the deletion that was confirmed as a de novo variant by Sanger sequencing (Fig 2). This heterozygous CACNA1A deletion brings about a ΔF1502 change in the CaV2.1 α1A channel subunit, previously described in association with both congenital ataxia and hemiplegic migraine [44].


A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of CaV2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca2+ Influx.

Bahamonde MI, Serra SA, Drechsel O, Rahman R, Marcé-Grau A, Prieto M, Ossowski S, Macaya A, Fernández-Fernández JM - PLoS ONE (2015)

De novo heterozygous CACNA1A deletion in congenital ataxia with cerebellar atrophy.(A) Pedigree of the affected individual carrying the de novo heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696675&req=5

pone.0146035.g002: De novo heterozygous CACNA1A deletion in congenital ataxia with cerebellar atrophy.(A) Pedigree of the affected individual carrying the de novo heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).
Mentions: Due to the large number of genes potentially causing cerebellar ataxia, we performed whole exome sequencing (WES) of a parent-child trio with an unknown cause of congenital ataxia in the child. Variant detection on the sequencing data using GATK [45] and ClinDel [47] yielded 115,818 and 12,753 variants, respectively. Variants affecting the protein sequence (nonsynonymous or splicing) were filtered based on possible inheritance modes, including de novo, recessive, compound heterozygous or X-linked. Variants coherent with one of the inheritance modes were further prioritized based on population allele frequency in EVS and 1000GP (i.e. rare variants with < 1% AF), damage potential based on PolyPhen-2, SIFT, Condel and CADD [49–52], evolutionary conservation based on phastCons [53] and finally sequence complexity and repetitiveness (e.g. variants in segmental duplications are removed), finally yielding a total of seven de novo, eight recessive and 15 compound heterozygous variants that are rare or novel and highly damaging. The affected 30 genes were subsequently subjected to additional prioritization using ENDEAVOUR [54], which has been trained on genes extracted from various disease gene resources [55,56]. The ENDEAVOUR prioritization highlighted a de novo triplet deletion in CACNA1A (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) as most likely candidate, as being part of the list of training genes. Furthermore, no other genes after filtering could be linked to the investigated phenotype. Manual review of the respective alignment data supports the presence of the deletion that was confirmed as a de novo variant by Sanger sequencing (Fig 2). This heterozygous CACNA1A deletion brings about a ΔF1502 change in the CaV2.1 α1A channel subunit, previously described in association with both congenital ataxia and hemiplegic migraine [44].

Bottom Line: ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels.ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance.Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum.

Show MeSH
Related in: MedlinePlus