T-type Ca2+ channels and aldosterone secretion

T型Ca2通道与醛固酮分泌

• 批准号: 7368001
• 负责人: PAULA Q BARRETT
• 金额: $ 32.47万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368001
• 关键词: Adrenal Glands; Affinity; Aldosterone; Angiotensin II; Binding; Biochemistry; Cells; Cellular biology; Chronic Kidney Failure; Disease Progression; Dopamine; Electrophysiology (science); Functional disorder; Heart failure; Hormones; Laboratories; Localized; Mediating; Molecular; Molecular Biology; Pathology; Peptides; Phosphotransferases; Physiological; Production; RNA Interference; Recruitment Activity; Regulation; Renin-Angiotensin-Aldosterone System; Resistance; Signal Transduction; Signaling Molecule; Site; Testing; Transmembrane Domain; Viral; Zona Glomerulosa; base; calmodulin-dependent protein kinase II; dimer; in vivo; tool; viral RNA; voltage

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.

描述(由申请人提供)：心力衰竭和慢性肾脏疾病都会导致神经激素的激活状态，从而加速它们的进展。这些病理生理的核心是肾素-血管紧张素系统的激活和醛固酮的产生。在球状带(AG)的醛固酮产生细胞中，醛固酮的产生依赖于钙离子，ALH的低电压激活(LVA)、T型、钙通道是钙电流的主要载体。我们的实验室已经发现，连接Alpha1H通道上跨膜结构域II和III的细胞内环(II-III环)是信号整合的重要中心。CaMKII使S1198磷酸化，导致门控的半激活电位超极化漂移，而GBbeta2ggamma2与高亲和力结合并独立抑制aIpha1H通道活动电压。我们验证了这样的假设：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，从而使相互的通道调节能够在功能上促进Ang II和多巴胺的生理作用，这两种激素对醛固酮的产生施加强大的反调节控制。 我们使用分子生物学、生物化学、细胞生物学和电生理学的工具来检验这一假说，具体目的如下：目标1：具体地说，我们将：(1.1)鉴定介导高亲和力CaMKII结合的Alpha1H II-III环上的残基，(1.2)确定这种结合是否在细胞刺激期间动态地将激酶定位于通道，(1.3)将多肽或CaMKII调节抗性通道引入肾上腺球状带细胞以干扰通道调节，并评价Ang II对醛固酮分泌的刺激作用。目的2：具体地说，我们将：(2.1)确定介导抑制Alpha1H全细胞通道活性和Alpha1H II-III环结合的GBeta2亚基上的关键残基，(2.2)确定Gbeta2亚基是否抑制摘除的斑块中的Ohh通道，(2.3)利用RNAi和病毒介导的将通道调节缺陷的Gbeta亚基输送到肾上腺带肾小球细胞来干扰通道调节，评价多巴胺对醛固酮分泌的抑制作用。