A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia

Abstract

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel’s loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.

A Common Kinetic Property of Mutations Linked to Episodic Ataxia Type 1 Studied in the Shaker Kv Channel

“…With this shift in conductance, also the activation, deactivation and inactivation were shifted to depolarized potentials. Such a shift of the conductance voltage relation to less polarized potentials had been previously reported for other EA1 mutations including R297T, F373V, R377F, G381D, V478A and F484S [ 8 , 9 , 10 , 11 , 12 ]. We should mention that the experiments were carried out at room temperature, as it is customary for Xenopus oocytes.…”

Kv1.1 Channelopathies: Pathophysiological Mechanisms and Therapeutic Approaches

“…Kv1.1 mutants associated with more severe EA1 phenotypes with long-lasting ataxia produce very small currents and often exert dominant-negative effects on co-expressed wild-type subunits (C185W in [62]; R324T in [74]; F414S in [55]; R417stop in [73]). The shift of the voltage dependence of activation towards positive potentials is a recurrent channel dysfunction of mutations identified in patients with typical EA1 symptoms (V404I in [57]; I177N in [75]; F184C in [33,76]; E283K in [64]; E325D in [77]; F303V in [78]). Altered kinetics of slow inactivation is another reported biophysical defect that may further reduce Kv1.1 channel availability (V408A in [19,76,77]).…”

“…These functional studies have been also critical to establish novel structure-function relationships in Kv1.1 channels. Indeed, the characterization of EA1 mutations demonstrated that: (i) residues in the S1 region facing the voltage-sensor domain contribute to Kv1.1 gating (I177N in [75]; F184C in [79]; C185W in [62]); (ii) amino acidic substitutions perturbing the interactions between residues of adjacent helices result in channels with reduced probability to open at physiological membrane potentials (E325D in S4-S5 linker in [77]; E283K in S3-S4 linker in [64]; F303V in S4 in [78]); (iii) impairment of the flexibility of S6 segment can affect open channel stability (V404I in [80]; V408A in [19,76]).…”

“…EA1 mutations can also affect the physiological modulation of Kv1.1 channels by intracellular and extracellular factors. In vitro, the EA1 mutation F184C sensitizes both homomeric Kv1.1 and heteromeric Kv1.1/Kv1.4 channel to extracellular Zn 2+ block implying that the Zn 2+ effects may be additional to the intrinsic gating defect caused by the mutation [33,78,83]. These in vitro findings suggest that similar functional alterations may occur in the brain of EA1 patients where Kv1.1, Kv1.2 and Kv1.4 subunits likely co-assemble and Zn 2+ is released, recapitulating different clinical phenotypes.…”

Identification of a New de Novo Mutation Underlying Regressive Episodic Ataxia Type I

“…EA1 is an autosomal dominant disorder associated with KCNA1 that codes the voltage-dependent K + channel Kv1.1. Indeed, several heterozygous missense mutations have been identified that resulted in different expression and gating defects in the Kv1.1 channel ( 1 , 8 – 16 ). The disease is distinguished by symptoms that include muscle twitching (myokymia) and an episodic loss of motor control, coordination, and balance that is associated with spastic head, arm, and leg muscle contractions.…”