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

在单通道分子水平上了解 BK 通道病的新方法

• 批准号: 10639690
• 负责人: KARL L MAGLEBY
• 金额: $ 43.94万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639690
• 关键词: Alleles; Ataxia; Atrophic; Biophysics; Categories; Cell membrane; Cells; Cerebrum; Complication; Congenital Abnormality; Coupling; Developmental Delay Disorders; Disease; Dominant-Negative Mutation; Electrophysiology (science); Epilepsy; Fragile X Syndrome; Frequencies; Genes; Genetic Diseases; Genetic Predisposition to Disease; Genetic Variation; Heterozygote; Hybrids; Individual; Intellectual functioning disability; Laboratories; Modeling; Molecular; Muscle hypotonia; Mutation; Patients; Phenotype; Physiological; Population; Potassium Channel; Property; Reporting; Severities; Syndrome; System; Techniques; Testing; Variant; arterial tortuosity; autism spectrum disorder; bone; clinical phenotype; expectation; gain of function; genetic variant; large-conductance calcium-activated potassium channels; loss of function; malformation; mutant; nervous system disorder; novel strategies; prevent; stoichiometry; voltage

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.

抽象的 在单通道分子水平上了解 BK 通道病的新方法。 KCNMA1 BK 钾通道的突变会产生多种通道病，包括癫痫、 运动障碍、自闭症、多种先天性畸形、智力障碍、发育迟缓、中轴 肌张力低下、共济失调、大脑和小脑萎缩、骨质增厚、动脉迂曲、畸形 综合症等。我们建议大多数旨在了解生物物理学基础的研究 这些 BK 通道病尚未研究主要通道；我们计划这样做。大多数 这些通道病发生在突变基因杂合的个体中。由于 BK 频道是 四聚体，由四个相似的亚基组成，突变亚基可以与野生型（WT）亚基组装形成 具有不同化学计量的通道的集合。绝大多数合奏频道 (88%) 将 是含有不同数量的突变亚基（从一到三个）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具有 通过在实验室表达系统中仅表达突变亚基来在实验室中进行研究。 假设亚基随机组装，纯突变通道不会占总通道数的 6% 以上 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，许多突变被 由于增强或功能的发现，被分为功能获得（GOF）或功能丧失（LOF）类别 分别基于同源四聚体突变通道，减少 BK 通道的激活。我们建议 忽略研究这些患者体内存在的完整通道类型导致了混乱和不准确的结果 通过没有认识到这些患者的大多数异常通道可能是杂种来对表型进行分类 它们构成了杂合性通道病中的大多数异常通道。在目标 1 中使用 结合包括单通道分析在内的电生理学技术，我们建议证明细胞 携带一个突变体和一个 WT KCNMA1 等位基因，表达一组由杂种主导的 BK 通道 由突变体和WT亚基组装而成的通道。在目标 2 中，我们将确定功能属性 和门控机制，以确定与 BK 通道病相关的主要通道如何形成 （如目标 1 中所确定）改变通道激活。在目标 3 中，我们将检验一些遗传变异的假设 BK 通道的数量导致亚基被截短，从而导致细胞中 BK 电流量减少 与 WT 亚基杂合。尽管这些变异基因在人群中传播，但并未被报道 杂合时引起神经系统疾病。尽管如此，这些突变仍需要研究，因为 BK 电流减少可能会赋予神经系统疾病潜在的遗传倾向。这些目标随后将 在所有表达通道类型的单通道分子水平上表征 BK 变体的作用， 提供了解通道病的关键信息。