A Genetic Approach to Identify New Regions Involved BK Channel Ca2+ Gating

识别涉及 BK 通道 Ca2 门控的新区域的遗传方法

• 批准号: 7275494
• 负责人: Brandon E Johnson
• 金额: $ 3.24万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7275494
• 关键词: Acetylcholine; Acetylcholinesterase Inhibitors; Affect; Aldicarb; Alleles; Binding; Biological Process; Biophysics; Caenorhabditis elegans; Calcium; Cardiac; Class; Conflict (Psychology); Cystic Fibrosis; Defect; Dependence; Disease; Epilepsy; Exons; Functional disorder; Genes; Genetic; Genetic Screening; Goals; Human; Inherited; Ion Channel; Lead; Lesion; Measures; Membrane; Membrane Potentials; Missense Mutation; Modeling; Molecular; Muscle; Mutate; Mutation; Nematoda; Nerve Degeneration; Neuromuscular Junction; Neurons; Paralysed; Pattern; Phenotype; Physiological; Potassium; Potassium Channel; Property; Protein Isoforms; Protein Region; Proteins; RNA Splicing; Recovery; Role; Signal Transduction; Synaptic Transmission; Transgenic Organisms; Variant; Work; Xenopus oocyte; cell type; deafness; large-conductance calcium-activated potassium channels; mutant; protein function; quantum; voltage

DESCRIPTION (provided by applicant): Ion channel dysfunction causes many inherited disease, including cystic fibrosis, cardiac dysfunction, neurodegeneration and deafness. A better understanding of how ion channels function will lead to better treatments for these diseases. The large-conductance, calcium-activated potassium (BK) channel is expressed in numerous cell types where it functions to repolarize membrane potential. A mutation in the human BK channel affects calcium activation and causes epilepsy. The molecular mechanism of calcium activation remains unknown. The goal of this study is to locate and evaluate regions of the protein that are important for BK channel activation. To discover such regions, we conducted a mutant screen in the nematode Caenorhabditis elegans. Thus far, this screen revealed twelve alleles of the slo-1 gene that encodes the BK channel and the first known splice variant-specific worm slo-1 mutant. This result indicates that biophysical differences may exist between BK channels isoforms and that these differences are physiologically relevant. Aim 1 proposes to examine slo-1 splice variants. First, we will examine the expression pattern of the slo-1 c splice variant. We will specifically determine if slo-1 c functions pre- or post- synaptically at the NMJ to determine if splice variants have distinct or overlapping functions. Finally, we will determine whether the biophysical properties of splice variants are distinct by measuring macroscopic currents from BK channel isoforms in inside-out patches from Xenopus oocytes containing many channels. Aim 2 proposes to sequence the remaining new BK channel alleles derived from our screen and to examine how these mutations affect channel biophysics in membrane patches. We predict that a subset of these slo-1 mutants will have defects in calcium sensitivity compared to wild-type channels. These results will be used to construct a model to evaluate how the mutated regions contribute to calcium activation and BK channel function. Ion channels are a class of proteins that function to generate electrical signals in the nervous sytem. A mutation in a human potassium channel causes epilepsy. We are examining how the structural features of this protein confer its biological function. We believe that the results of our work will contribute to the discovery of better treatments for epilepsy and other ion channel-related diseases.

描述（由申请人提供）：离子通道功能障碍会引起许多遗传疾病，包括囊性纤维化，心脏功能障碍，神经退行性变性和耳聋。更好地了解离子通道功能将为这些疾病提供更好的治疗方法。大传统的，钙激活的钾（BK）通道在许多细胞类型中表达，在许多细胞类型中，其功能可重复膜电位。人BK通道中的突变会影响钙的激活并引起癫痫。钙激活的分子机制仍然未知。这项研究的目的是定位和评估对BK通道激活很重要的蛋白质区域。为了发现这样的区域，我们在线虫秀丽隐杆线虫中进行了突变筛查。到目前为止，该屏幕揭示了编码BK通道的SLO-1基因的十二个等位基因和第一个已知的剪接变体特异性蠕虫SLO-1突变体。该结果表明，BK通道同工型之间可能存在生物物理差异，并且这些差异在生理上是相关的。 AIM 1建议检查SLO-1剪接变体。首先，我们将检查SLO-1 C剪接变体的表达模式。我们将明确确定SLO-1 C函数是否在NMJ处进行突触前或后突触，以确定剪接变体是否具有不同的或重叠的函数。最后，我们将确定剪接变体的生物物理特性是否通过测量来自包含许多通道的Xenopus卵母细胞的内部斑块中的BK通道同工型的宏观电流来不同。 AIM 2提出，将剩余的新的BK通道等位基因序列序列，并检查这些突变如何影响膜斑块中的通道生物物理学。我们预测，与野生型通道相比，这些SLO-1突变体的子集将具有钙敏感性缺陷。这些结果将用于构建模型，以评估突变区域如何促进钙激活和BK通道功能。离子通道是一类蛋白质，可在神经系统中产生电信号。人类钾通道中的突变会引起癫痫。我们正在研究该蛋白质的结构特征如何赋予其生物学功能。我们认为，我们的工作结果将有助于发现癫痫和其他与离子渠道相关疾病的更好治疗方法。