Phosphorylation as a Determinant of BK Channel Expression and Localization

描述(申请人提供)：BK通道是神经元信号的基本组成部分，通过影响神经元的兴奋性和突触传递。这一建议旨在确定控制哺乳动物海马区BK通道表达和定位的基本机制。使用新的、最先进的质谱学方法，我们在确定从大鼠脑中纯化的BK通道的初级或BK1亚单位上的体内磷酸化位点方面取得了很大进展。我们发现，大多数体内的磷酸化位点都是Pro相关的pSer，并且符合含有pSer结合模块的蛋白质的共识结合位点。此外，这些位点可能被Pro-DKs(ProDKs)磷酸化，ProDKs在神经元功能中的重要性以及作为新疗法的靶点刚刚被认识到。这些数据为研究BK通道上真实且明确识别的脑内亚磷酸盐在调节其在海马区的表达水平和亚细胞定位中的作用提供了第一次机会。我们将测试这一建议的总体假设，即这些位置对作用于天然BK通道的ProDK介导的神经元功能和可塑性至关重要。在目标1中，我们将通过检查改变BK1磷酸化状态的干预的效果来实现这一点。我们将在体内突变已确定的ProDK磷酸化位点，并将干预异源细胞和海马神经元中ProDK的表达水平，并确定对BK通道表达的影响。在目标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BK通道的生理和病理调控产生重要的影响，BK通道是哺乳动物海马区神经元兴奋性和突触传递的关键调节因子。公共卫生相关性：这项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节酶，它们是开发神经和精神疾病新疗法的重要目标。

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.