EA1突變對於人類鉀離子通道hKv1.1及hKv1.4生物物理特性之影響

Abstract

第一型陣發性運動失調症 (Episodic ataxia type 1, EA1) 是一種體染色體顯性遺傳的神經性疾病。EA1是由於人類第十二對染色體 (12p13) 上的KCNA1基因突變所致，而KCNA1基因負責轉錄人類的電位控制鉀離子通道Kv1.1 (hKv1.1)。目前為止已經在EA1病人的KCNA1基因上發現了20種突變，而本篇研究主要想探討I262T和R292K兩種EA1突變是否會改變hKv1.1和hKv1.4的生物物理性質，並進而推測其神經生理意義。 我們將各種cRNA表達在非洲爪蟾的蛙卵上，再利用雙電極電位箝制技術來記錄電流。首先第一部分是針對hKv1.1 monomer (WT)，I262T會造成通道的電流表現量降低，R292K則不影響電流量。兩種突變皆會使開啓速率變慢、關閉速率變快及造成活化曲線往右偏移。I262T使得通道進入不活化態的速率變慢，但從不活化態回復的速率則會變快；R292K也造成不活化速率變慢。I262T和R292K都會造成不活化曲線往右偏移。第二部分是針對hKv1.1-Kv1.1 dimer (WT-WT dimer)，I262T和R292K並不會影響dimer的表現量，但兩種突變依然會使開啓速率變慢、關閉速率變快。同樣地，I262T會造成活化曲線往右偏移。I262T和R292K會造成通道進入不活化態的速率變慢。而兩者皆不改變通道從不活化態回復的速率。I262T和R292K也會造成不活化曲線往右偏移但兩者偏移的電位範圍和斜率都差不多。我們也將正常型和突變型的hKv1.1共同表現在細胞上 (WT + I262T；WT + R292K)，I262T會造成電流表現量降低，而R292K依然不影響電流表現量；其活化曲線往右偏移的電位範圍和斜率皆和dimer差不多。第三部分則是針對hKv1.4與hKv1.1之相互作用，無論是共同表現 (hKv1.4 + WT) 或是hKv1.4-Kv1.1 dimer (hKv1.4-WT dimer)，I262T都會造成電流表現量降低，而R292K則不改變電流表現量；兩種突變也都會使不活化曲線往右偏移，但兩者皆不改變hKv1.4-WT dimer不活化的程度、不活化速率和通道從不活化態回復的速率。當hKv1.4-WT dimer和hKvbeta1.1組合在一起時，I262T和R292K使通道在較低電位時不活化程度變小但同樣不改變不活化速率。兩種突變仍使不活化曲線往右偏移也讓通道從不活化態回復的速率變快。 由以上的實驗結果發現，I262T和R292K對於神經細胞上不同的通道組合會有相反的放電模式，進而造成了EA1病人在行為表現上出現異常。

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder. EA1 is caused by point mutations in the gene KCNA1 on chromosome 12p13, which encodes the human voltage-gated potassium channel Kv1.1 (hKv1.1). To date, 20 mutations in KCNA1 gene have been reported. In this study, we want to investigate whether two EA1 mutations (I262T;R292K) alter the biophysical properties of hKv1.1 and hKv1.4, and further to speculate the neurophysiological significance. All kinds of cRNAs expressed in Xenopus laevis oocytes were studied by two-electrode voltage clamp. Here we showed that I262T reduced the current amplitude and R292K didn’t. Both EA1 mutations shifted the voltage dependence of activation to more depolarized potentials. They also slowed the activation kinetics and accelerated the deactivation kinetics of the hKv1.1. Both EA1 mutations shifted the voltage dependence of inactivation to more depolarized potentials and decreased the rate of N-type inactivation. Furthermore, I262T increased the recovery rate from inactivation. Next, we examined hKv1.1-mutant heterotetramer (WT-mutant dimer). Both EA1 mutations slowed the activation kinetics, accelerated the deactivation kinetics and decreased the rate of N-type inactivation, but they didn’t alter the current amplitude and the recovery rate from inactivation. I262T shifted the voltage dependence of activation to more depolarized potential and both EA1 mutations shifted the voltage dependence of inactivation to more depolarized potentials. To examine the effects of expressing each EA subunit with wild-type subunit, equal amounts of EA and wild-type cRNAs were coinjected. We found that I262T reduced the current amplitude and R292K didn’t. Both EA1 mutations shifted the voltage dependence of activation to more depolarized potentials. Finally, we examined heteromeric channels containing a fixed stoichiometry of two wild type hKv1.4 and two mutant hKv1.1 (hKv1.4-mutant heterotetramer). I262T reduced the current amplitude and R292K didn’t. Both EA1 mutations positively shifted the voltage dependence of inactivation and they didn’t alter the amount of N-type inactivation, the inactivation rate and the recovery rate from inactivation. When hKv1.4-WT heterotetramer coexpressed the hKvbeta1.1, the hKvbeta1.1 subunits would increase the amount of N-type inactivation of hKv1.4-WT dimer. The two EA1 mutations affected the hKv1.4-WT dimer + hKvbeta1.1 by positively shifting the voltage dependence of inactivation and increasing the recovery rate from inactivation, but they didn’t alter the inactivation rate. In conclusion, if homomeric hKv1.1 and heteromeric hKv1.1 ⁄ hKv1.4 ⁄ hKvbeta1.1 coexisted in the same neuron then the opposite biophysical effects of the mutations on these channels probably impaired the distinct nerve cell property and caused the defects of body movements in EA1 patients.