T-type Ca2+ channels and aldosterone secretion

描述（由申请人提供）：心力衰竭和慢性肾脏疾病都会诱发神经激素激活状态，从而加速其进展。这些病理生理学的核心是肾素-血管紧张素系统的激活和醛固酮的产生。醛固酮的产生是ca2 +依赖和alH低压激活（LVA）的，t型ca2 +通道是肾小球带（AG）醛固酮产生细胞中ca2 +电流的主要载体。本实验室在alpha1H通道上发现连接跨膜结构域II和III的胞内环（II-III环）是信号整合的重要中心。CaMKII磷酸化S1198，诱导门控半激活电位的超极化移位，gbbeta2gamma2高亲和力结合，独立抑制aIpha1H通道活性电压。我们验证了这样的假设，即在细胞激活过程中，II- iii环选择性地、高亲和力地招募这些信号分子，从而使相互通道调节在功能上有助于Ang II和多巴胺的生理活动，这两种激素对醛固酮的产生施加强烈的反调节控制。

DESCRIPTION (provided by applicant): Both heart failure and chronic renal disease induces a state of neurohormonal activation that hastens their progression. Central to these pathophysiologies is the activation of the Renin-Angiotensin system and aldosterone production. Aldosterone production is Ca 2+ dependent and alH low-voltage-activated (LVA), T-type, Ca 2+ channels are the major carriers of Ca 2+ current in the aldosterone producing cell of the zona glomerulosa (AG). Our laboratory has identified the intracellular loop connecting transmembrane domains II and III (II-III loop) on alpha1H channels as an important center for signal integration. CaMKII phosphorylates S1198 to induce a hyperpolarizing shift in the half-activation potential for gating, and GBbeta2ggamma2binds with high-affinity and inhibits aIpha1H channel activity voltage independently. We test the hypothesis that during cell activation the II-III loop recruits these signaling molecules selectively and with high-affinity and thus enables reciprocal channel regulation to contribute functionally to the physiologically actions of Ang II and dopamine, two hormones that exert strong counter-regulatory control of aldosterone production. We use tools of molecular biology, biochemistry, cell biology and electrophysiology to test this hypothesis in the following specific aims: Aim 1: Specifically we will: (1.1) identify the residues on the alpha1H II-III loop that mediate high affinity CaMKII binding, (1.2) determine if this binding dynamically localizes the kinase to the channel during cell stimulation, (1.3) introduce peptides or CaMKII-regulation resistant channels to adrenal zona glomerulosa cells to perturb channel regulation and evaluate the stimulation of aldosterone secretion by Ang II. Aim 2: Specifically we will: (2.1) identify the critical residues on GBeta2 subunits that mediate inhibition of alpha1H whole-cell channel activity and alpha1H II-III loop binding, (2.2) establish if Gbeta2 subunits inhibit ohH channels in the excised patch, (2.3) use RNAi and viral-mediated delivery of channel regulation-deficient Gbeta subunits to cells of the adrenal zona glomerulosa to disrupt channel regulation and evaluate the inhibition of aldosterone secretion by dopamine.