New approaches to understanding BK channelopathies at the molecular level of single channels

摘要 在单通道分子水平上理解BK通道病的新方法。 KCNMA1 BK钾通道的突变会导致广泛的通道病变，包括癫痫， 运动障碍、自闭症、多发性先天畸形、智力残疾、发育迟缓、轴性 低眼压、共济失调、脑和小脑萎缩、骨质增厚、动脉扭曲、畸形 综合症，以及其他。我们认为，大多数试图了解糖尿病的生物物理基础的研究 这些BK通道病一直没有研究主要的通道；我们计划这样做。大多数人 这些通道病发生在突变基因杂合子的个体中。由于BK频道 四聚体，由四个相似的亚基组成，突变的亚基可以与野生型(WT)亚基组装形成 具有不同化学计量比的通道的集合。绝大多数(88%)的合奏频道将 是含有不同数量的突变亚基(从1个到3个)的异四聚体杂交通道。然而， 这些混合通道通常被忽视，在通道病患者中发现的突变通常 在实验室中通过在实验室表达系统中仅表达突变的亚基来进行研究。 假设亚基的随机组装，纯突变通道不会超过6%的 在杂合子患者的细胞中发现的通道。基于这些不完整的研究，许多突变是 根据增强型或非增强型 BK通道的激活减少，分别基于同源四聚体突变通道。我们建议 忽略了对这些患者存在的所有通道类型的研究，导致了一种混乱和不准确的 由于没有认识到这些患者中最异常的通道可能是杂交体而进行的表型分类 它们构成了杂合性通道病中异常通道的大部分。在目标1中使用 结合包括单通道分析在内的电生理技术，我们建议证明细胞 携带一个突变体和一个WT KCNMA1等位基因表达由杂交主导的BK通道集合 通道由突变体和WT亚基组装而成。在目标2中，我们将确定函数性质 以及确定与BK通道病相关的主要通道如何形成的门控机制 (如目标1中所确定的)更改通道激活。在目标3中，我们将检验这样的假设：某些遗传变异 导致BK通道亚基被截断，从而导致细胞内BK电流的减少 WT亚基为杂合子。虽然这些变异基因在人群中传播，但尚未有报道。 当杂合子时会导致神经系统疾病。然而，这些突变需要研究，因为 BK电流的减少可能会使神经疾病的遗传倾向变得隐秘。届时，这些目标将 在所有表达的通道类型的单个通道的分子水平上表征BK变体的作用， 提供了解通道病的关键信息。

ABSTRACT New approaches to understanding BK channelopathies at the molecular level of single channels. Mutations in KCNMA1 BK potassium channels produce a wide range of channelopathies which include epilepsy, dyskinesis, autism, multiple congenital abnormalities, intellectual disability, developmental delay, axial hypotonia, ataxia, cerebral and cerebellar atrophy, bone thickening, tortuosity of arteries, malformation syndrome, and others. We suggest that the majority of studies that seek to understand the biophysical basis of these BK channelopathies have not been studying the predominant channels; we plan to do so. The majority of these channelopathies arise in individuals who are heterozygous for the mutant gene. Since BK channels are tetrameric, composed of four like subunits, mutant subunits could assemble with wild-type (WT) subunits to form an ensemble of channels with different stoichiometries. The vast majority of the ensemble channels (88%) would be heterotetrameric hybrid channels containing a varied number of mutant subunits (from one to three). However, these hybrid channels are typically overlooked, and the mutations found in channelopathy patients have usually been studied in the laboratory by expressing only the mutant subunit in a laboratory expression system. Assuming random assembly of subunits, the purely mutant channels would not represent more than 6% of the channels found in the cells of a heterozygous patient. Based on these incomplete studies many mutations were categorized into gain-of-function (GOF) or loss-of-function (LOF) categories due to the findings of enhanced or reduced activation of BK channels, respectively based on homotetrameric mutant channels. We suggest that the omission of studying the full palette of channel types present in these patients has led to a chaotic and inaccurate categorization of phenotypes by not recognizing that most aberrant channels in these patients may be hybrids which constitute the majority of the aberrant channels in heterozygous channelopathies. In Aim 1 using a combination of electrophysiological techniques including single channel analysis, we propose to show that a cell carrying one mutant and one WT KCNMA1 allele expresses an ensemble of BK channels dominated by hybrid channels assembled from both mutant and WT subunits. In Aim 2 we will determine the functional properties and gating mechanisms to determine how the predominant channel forms associated with BK channelopathies (as determined in Aim 1) alter channel activation. In Aim 3 we will test the hypothesis that some genetic variants of BK channels result in a truncated subunit that leads to a reduced amount of BK current in cells when heterozygous with WT subunits. Although these variant genes circulate in the population they are not reported to cause neurological disease when heterozygous. Nevertheless, these mutations need to be studied because reduced BK currents could confer a furtive genetic pre-disposition to neurological disease. These aims will then characterize the actions of BK variants at the molecular level of single channels for all expressed channel types, providing information key towards understanding the channelopathies.