KCNC3: phenotype, mutations, channel biophysics-a study of 260 familial ataxia patients

Abstract

We recently identified KCNC3, encoding the Kv3.3 voltage-gated potassium channel, as the gene mutated in SCA13. One g.10684G>A (p.Arg420His) mutation caused late-onset ataxia resulting in a nonfunctional channel subunit with dominant-negative properties. A French early-onset pedigree with mild mental retardation segregated a g.10767T>C (p.Phe448Leu) mutation. This mutation changed the relative stability of the channel's open conformation. Coding exons were amplified and sequenced in 260 autosomal-dominant ataxia index cases of European descent. Functional analyses were performed using expression in Xenopus oocytes. The previously identified p.Arg420His mutation occurred in three families with late-onset ataxia. A novel mutation g.10693G>A (p.Arg423His) was identified in two families with early-onset. In one pedigree, a novel g.10522G>A (p.Arg366His) sequence variant was seen in one index case but did not segregate with affected status in the respective family. In a heterologous expression system, the p.Arg423His mutation exhibited dominant-negative properties. The p.Arg420His mutation, which results in a nonfunctional channel subunit, was recurrent and associated with late-onset progressive ataxia. In two families the p.Arg423His mutation was associated with early-onset slow-progressive ataxia. Despite a phenotype reminiscent of the p.Phe448Leu mutation, segregating in a large early-onset French pedigree, the p.Arg423His mutation resulted in a nonfunctional subunit with a strong dominant-negative effect.

Figure 1

Cerebellar atrophy in individual MR 3-3 with an p.Arg420His mutation. A) T1-weighted axial image of posterior fossa. B) T2-weighted midsaggital image, note lack of brainstem atrophy.

Figure 2

Dominant negative effects of KCNC3 mutations. Ai and Bi: p.Arg423His (A) or p.Arg366His (B) was expressed in Xenopus oocytes for functional analysis. Currents were evoked by stepping from −90 mV to voltages ranging from −80 to +70 mV in 10 mV increments. Aii and Bii: Shown are representative current traces evoked by stepping from −90 mV to +60 mV for wild-type Kv3.3 expressed alone or in the presence of p.Arg423His or p.Arg366His at the indicated ratios. Aiii and Biii: Normalized peak current amplitudes measured at +60 mV are shown for wild-type expressed alone or with p.Arg423His or p.Arg366His at the indicated ratios. For comparison, current amplitude values for p.Arg423His or p.Arg366His expressed alone or for uninjected oocytes (Uninj) are also provided. Values are provided as SEM ± mean, n=4–10. Statistical significance was tested by one-way analysis of variance (ANOVA). P< 0.05: *, significantly different from 1:0; **, significantly different from 1:1, ***, significantly different from 1:4. Aiv and Biv: Representative current traces recorded at +60 mV of wild type expressed alone (dotted traces) and co-expression of p.Arg423His (Aiv) or p.Arg366His (Biv) at a 1:1 ratio with wild type (solid traces) have been scaled and overlaid for comparison.