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.
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