title: 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.
authors.affiliation: Department of Physiology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Jenny
authors.initials: J
authors.lastname: van der Wijst
authors.forename: Bob
authors.initials: B
authors.lastname: Glaudemans
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Anil V
authors.initials: AV
authors.lastname: Nair
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Forst
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001216:Asparagine / Q000378:metabolism; D002460:Cell Line; D005799:Genes, Dominant / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D051662:Kv1.1 Potassium Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary; D015499:Renal Tubular Transport, Inborn Errors / Q000235:genetics*; D013499:Surface Properties; D013997:Time Factors
chemical_list: C496946:KCNA1 protein, human; D050505:Mutant Proteins; D051662:Kv1.1 Potassium Channel; D001216:Asparagine