Phosphorylation as a Determinant of BK Channel Expression and Localization

描述（申请人提供）：BK通道是神经元信号传导的基本组成部分，通过影响神经元的兴奋性和突触传递。本研究旨在确定哺乳动物海马中BK通道表达和定位的基本机制。使用新颖的、最先进的质谱方法，我们在确定从大鼠脑中纯化的BK通道的初级或BK1亚基上的体内磷酸化位点方面取得了重大进展。我们发现大多数体内磷酸化位点都是亲相关的pSer，并且与含有pSer结合模块的蛋白质的一致结合位点一致。此外，这些位点很可能被脯氨酸定向激酶（proline- directed kinase, ProDKs）磷酸化，而脯氨酸定向激酶在神经元功能和作为新疗法靶点方面的重要性目前正在得到重视。这些数据提供了第一个机会来研究BK通道上的真实和明确的体内脑磷酸化位点在控制其表达水平和海马亚细胞定位中的作用。我们将测试这一提议的整体假设，即这些位点对神经元功能和可塑性至关重要，这些功能和可塑性是由作用于天然BK通道的prodk介导的。在目的1中，我们将通过检查改变BK1磷酸化状态的干预措施的影响来实现这一点。我们将突变体内鉴定的ProDK磷酸化位点，并将干预ProDK在异源细胞和海马神经元中的表达水平，并确定对BK通道表达的影响。在Aim 2中，我们将使用类似的方法来确定这些磷酸化位点在BK通道极化定位中的作用。这些研究将对哺乳动物海马神经元兴奋性和突触传递的关键调节因子BK通道的生理和病理调节提供重要的见解。公共卫生相关性：本研究旨在更好地了解控制大脑功能的基本机制。它侧重于神经元离子通道及其调控酶，这是开发神经和精神疾病新疗法的重要靶点。

DESCRIPTION (provided by applicant): BK channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of BK channels in mammalian hippocampus. Using novel, state-of- the art mass spectrometric approaches we have made great inroads in defining the in vivo phosphorylation sites on the primary or BK1 subunit of BK channels purified from rat brain. We find that most in vivo phosphorylation sites are Pro-associated pSer and conform to consensus binding sites for proteins containing pSer-binding modules. Moreover, these sites are likely phosphorylated by proline- directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on BK channels in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native BK channels. In aim 1 we will accomplish this by examining the effects of interventions that alter the phosphorylation state of BK1. We will mutate identified in vivo ProDK phosphorylation sites, and will intervene in ProDK expression levels in heterologous cells and hippocampal neurons, and determine effects on BK channel expression. In Aim 2 we will use similar approaches to determine the role of these phosphorylation sites in polarized localization of BK channels. These studies will yield important insights into the physiological and pathological regulation of BK channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for neurological and psychiatric disorders.