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.

摘要 在单通道分子水平上理解BK通道病的新方法。 KCNMA 1 BK钾通道的突变产生广泛的通道病，包括癫痫， 运动障碍、自闭症、多种先天性异常、智力残疾、发育迟缓、轴性 张力减退、共济失调、大脑和小脑萎缩、骨增厚、动脉迂曲、畸形 综合症，以及其他。我们建议大多数寻求了解生物物理基础的研究 这些BK通道病还没有研究主要的通道;我们计划这样做。大多数 这些通道病出现在突变基因杂合的个体中。由于BK通道是 四聚体，由四个类似的亚基组成，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。绝大多数的合奏频道（88%）将 可以是含有不同数量的突变亚基（从一个到三个）的异源四聚体杂交通道。然而，在这方面， 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 通过在实验室表达系统中仅表达突变亚基，在实验室中进行了研究。 假设亚基的随机组装，纯突变通道将不代表超过6%的亚基。 在杂合子患者的细胞中发现的通道。根据这些不完整的研究， 由于增强或缺失的发现，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体通道的BK通道的激活减少。我们建议 遗漏研究这些患者中存在的通道类型的全部调色板已经导致了对这些患者的混乱和不准确的分析。 通过不认识这些患者中的大多数异常通道可能是杂合体来分类表型 其构成杂合性通道病中的大多数异常通道。在目标1中，使用 结合电生理技术，包括单通道分析，我们建议表明，细胞 携带一个突变型和一个野生型KCNMA 1等位基因的人表达由杂合型占主导地位的BK通道集合体， 由突变和WT亚基组装的通道。在目标2中，我们将确定函数属性 和门控机制，以确定与BK通道病相关的主要通道如何形成 (as目标1中确定的）改变通道激活。在目标3中，我们将测试一些遗传变异的假设， BK通道的减少导致一个截短的亚基，导致细胞中BK电流的减少， 与WT亚基杂合。虽然这些变异基因在人群中传播，但没有报道 导致神经系统疾病然而，这些突变需要研究，因为 BK电流的减少可能赋予神经系统疾病隐秘的遗传倾向。这些目标将 在所有表达的通道类型的单通道分子水平上表征BK变体的作用， 提供了了解通道病的关键信息。