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Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

ABSTRACT: Mutations in the voltage-gated K(+) channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936-942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, -41.5 +/- 1.6, -45.5 +/- 2.0, -50.5 +/- 1.9, and -33.8 +/- 1.3 mV to -29.4 +/- 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 +/- 0.2 ms) and N255V (5.2 +/- 0.3 ms), and the hydrophilic mutant N255T (9.8 +/- 0.4 ms) in comparison with wild type (13.0 +/- 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted approximately 13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.

SUBMITTER: van der Wijst J

PROVIDER: S-EPMC2804162 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

van der Wijst Jenny J Glaudemans Bob B Venselaar Hanka H Nair Anil V AV Forst Anna-Lena AL Hoenderop Joost G J JG Bindels René J M RJ

The Journal of biological chemistry 20091110 1

Mutations in the voltage-gated K(+) channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936-942). A conserved asparagine at position 255 in the ...[more]

PMID: 19903818

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Project description:IMPORTANCE:The family of genes implicated in hereditary spastic paraplegias (HSPs) is quickly expanding, mostly owing to the widespread availability of next-generation DNA sequencing methods. Nevertheless, a genetic diagnosis remains unavailable for many patients. OBJECTIVE:To identify the genetic cause for a novel form of pure autosomal dominant HSP. DESIGN, SETTING, AND PARTICIPANTS:We examined and followed up with a family presenting to a tertiary referral center for evaluation of HSP for a decade until August 2014. Whole-exome sequencing was performed in 4 patients from the same family and was integrated with linkage analysis. Sanger sequencing was used to confirm the presence of the candidate variant in the remaining affected and unaffected members of the family and screen the additional patients with HSP. Five affected and 6 unaffected participants from a 3-generation family with pure adult-onset autosomal dominant HSP of unknown genetic origin were included. Additionally, 163 unrelated participants with pure HSP of unknown genetic cause were screened. MAIN OUTCOME AND MEASURE:Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene. RESULTS:We identified the nucleotide substitution c.109C>T in exon 3 of CPT1C, which determined the base substitution of an evolutionarily conserved Cys residue for an Arg in the gene product. This variant strictly cosegregated with the disease phenotype and was absent in online single-nucleotide polymorphism databases and in 712 additional exomes of control participants. We showed that CPT1C, which localizes to the endoplasmic reticulum, is expressed in motor neurons and interacts with atlastin-1, an endoplasmic reticulum protein encoded by the ATL1 gene known to be mutated in pure HSPs. The mutation, as indicated by nuclear magnetic resonance spectroscopy studies, alters the protein conformation and reduces the mean (SD) number (213.0 [46.99] vs 81.9 [14.2]; P <?.01) and size (0.29 [0.01] vs 0.26 [0.01]; P <?.05) of lipid droplets on overexpression in cells. We also observed a reduction of mean (SD) lipid droplets in primary cortical neurons isolated from Cpt1c-/- mice as compared with wild-type mice (1.0 [0.12] vs 0.44 [0.05]; P <?.001), suggesting a dominant negative mechanism for the mutation. CONCLUSIONS AND RELEVANCE:This study expands the genetics of autosomal dominant HSP and is the first, to our knowledge, to link mutation in CPT1C with a human disease. The association of the CPT1C mutation with changes in lipid droplet biogenesis supports a role for altered lipid-mediated signal transduction in HSP pathogenesis.

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Dataset Information

Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

Publications

Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

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