T-type Ca2+ channels and aldosterone secretion

T型Ca2通道与醛固酮分泌

• 批准号: 7184349
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7184349
• 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均诱导神经激素激活状态，加速其进展。这些病理生理学的中心是激活肾素-血管紧张素系统和醛固酮产生。醛固酮的产生是钙依赖性的，肾小球醛固酮产生细胞（AG）中alH低压激活（LVA）的T型钙通道是Ca 2+电流的主要载体。我们的实验室已经确定了连接α 1H通道上跨膜结构域II和III的细胞内环（II-III环）作为信号整合的重要中心。CaMKII磷酸化S1198以诱导门控半激活电位的超极化偏移，而GB β 2 ggamma 2以高亲和力结合并独立地抑制aIpha 1 H通道活性电压。我们测试的假设，在细胞活化过程中的II-III环招募这些信号分子选择性和高亲和力，从而使相互通道调节功能有助于血管紧张素II和多巴胺，两种激素，发挥强大的反调节控制醛固酮的生产的生理作用。 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测试这一假设，具体目标如下：目标1：具体来说，我们将：（1.1）鉴定α 1 H II-III环上介导高亲和力CaMK II结合的残基，（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）将肽或CaMK II调节抗性通道引入肾上腺髓质肾小球细胞以扰乱通道调节并评估Ang II对醛固酮分泌的刺激。目标二：具体而言，我们将：（2.1）鉴定介导α 1 H全细胞通道活性和α 1 H II-III环结合抑制的G β 2亚基上的关键残基，（2.2）确定G β 2亚基是否抑制切除的贴片中的ohH通道，（2.3）使用RNAi和病毒介导的通道调节递送-缺乏G β亚单位的肾上腺肾小球细胞破坏通道调节和评价多巴胺对醛固酮分泌的抑制。