A novel KCND3 variant in the N-terminus impairs the ionic current of Kv4.3 and is associated with SCA19/22

Abstract

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of autosomal dominant movement disorders. Among the SCAs associated with impaired ion channel function, SCA19/22 is caused by pathogenic variants in KCND3, which encodes the voltage-gated potassium channel Kv4.3. SCA19/22 is clinically characterized by ataxia, dysarthria and oculomotor dysfunction in combination with other signs and symptoms, including mild cognitive impairment, peripheral neuropathy and pyramidal signs. The known KCND3 pathogenic variants are localized either in the transmembrane segments, the connecting loops, or the C-terminal region of Kv4.3. We have identified a novel pathogenic variant, c.455A>G (p.D152G), localized in the N-terminus of Kv4.3. It is located in the immediate neighbourhood of the T1 domain, which is responsible for multimerization with the β-subunit KChIP2b and thus for the formation of functional heterooctamers. Electrophysiological studies showed that p.D152G does not affect channel gating, but reduces the ionic current in Kv4.3, even though the variant is not located in the transmembrane domains. Impaired channel trafficking to the plasma membrane may contribute to this effect. In a patient with a clinical picture corresponding to SCA19/22, p.D152G is the first pathogenic variant in the N-terminus of Kv4.3 to be described to date with an effect on ion channel activity.

Keywords: KCND3; Kv4.3; SCA19/22; ataxia; electrophysiology; movement disorder; neuropathology.

FIGURE 1

(A) Pedigree of the family of the index patient. Black symbols indicate affected probands. The index patient is marked by an arrow. (B) Electropherograms of KCND3 sequences of the index patient and a control. The A>G transition at c.455 is indicated by an arrow. (C) Amino acid sequence alignments Kv4.3 orthologs. Name of species are given on the left. Amino acid mutated in the index patient is evolutionarily highly conserved and is highlighted in red. Non‐conserved amino acid residues are shown in green. H., Homo; G., Gorilla; P., Pan; M., Mus; R., Rattus; E.; Equus; F., Felis; X., Xenopus; D., Danio. (D) Schematic illustration of Kv4.3. S1 to S6 represent the transmembrane domains, while S4 is the voltage sensor. Amino acid changes that are associated with SCA19/22 are shown in orange. Changes that are associated with a cardiac phenotype (Brugada syndrome, sudden unexplained death syndrome, early repolarization syndrome, atrial fibrillation) are indicated in blue. Green indicates variants found in both patients with SCA19/22 and those with cardiac disease, or in patients with combined symptoms. The novel amino acid change p.D152G is located in the N‐terminus and is marked in red.

FIGURE 2

Analysis of Kv4.3 ion channel function as a homopolymer and as a heteropolymer with KChIP2b. (A) Current measurements of Kv4.3 WT, Kv4.3 p.D152G, and Kv4.3 WT/p.D152G (molar ratio 1:1). The number of independent experiments is given in parentheses. (B) Current measurements of Kv4.3 WT, Kv4.3 p.D152G, and Kv4.3 WT/p.D152G co‐expressed with KChIP2b (molar ratio 1:1). Number of independent experiments is given in parentheses. (C, D) Peak current amplitude of WT and variant channels in mean and S.E.M. Currents at 100 mV were tested with two‐tailed unpaired t‐test (*p < 0.05). (E) Normalized conductance (G/G_max) of Kv4.3 channels alone, and (F) with KChIP2b.

FIGURE 3

Inactivation kinetics of Kv4.3 with and without KChIP2b. (A) Current measurements of Kv4.3 WT, Kv4.3 p.D152G, and Kv4.3 WT/p.D152G (molar ratio 1:1). The number of independent experiments is given in parentheses. (B) Current measurements of Kv4.3 co‐expressed with KChIP2b (molar ratio 1:1). Number of independent experiments is given in parentheses. (C, D) Peak currents were normalized to I_max and leak current (I₀) of their Boltzmann fits (I/I_max) and plotted as a function of inactivation pulse voltage. (C) I/I_max of channels alone, and (D) with KChIP2b.

FIGURE 4

Recovery from inactivation kinetics of Kv4.3 with and without KChIP2b. (A) Current measurements of Kv4.3 WT, Kv4.3 p.D152G, and Kv4.3 WT/p.D152G (molar ratio 1:1). The number of independent experiments is given in parentheses. (B) Current measurements of Kv4.3 co‐expressed with KChIP2b (molar ratio 1:1). The number of independent experiments is given in parentheses. (C, D) Fits of peak current were normalized (I/I_max) and plotted as a function of interpulse interval with logarithmic scale. (C) Fits of channels alone, and (D) with KChIP2b.