Vascular Cav1.2 channels are the predominant source of Ca2+ entry in arterial myocytes. Consequently, these channels play a critical role for a wide variety of arterial functions, including excitation-contraction (EC) and excitation-transcription (ET) coupling. We have recently identified a new gating modality of Cav1.2 channels where a small subpopulation of these channels can gate in unison (i.e. coupled gating). The lack of a comprehensive mechanistic understanding of this gating modality or its functional implications in the regulation of arterial tone and blood pressure in health and disease represents major gaps in knowledge. This proposal aims to investigate the structural requirements and physiological consequences whereby dynamics of the ubiquitous Ca2+ sensor and regulatory molecule calmodulin (CaM) within the Cav1.2 channel complex underlies the coupling between these channels. To accomplish this, we are testing the novel central hypothesis that CaM serves as a Cav1.2 coupling tuner in response to changes in cytosolic Ca2+, and activation of PKA and PKCa, which are key molecules regulating Cav1.2 in arterial myocytes and elsewhere. In this model, AKAP150 serves as a hub for local channel regulation by anchored PKA, PKCa and calcineurin, and as the bond that facilitates coupling between adjacent channels. This central hypothesis has been formulated on the basis of strong preliminary data, and will be tested using a logical experimental progression that takes advantage of approaches well-established in the PI's or collaborators' labs such as heterologous expression systems, optogenetics, fluorescent biosensors, molecular biology, electrophysiology, confocal and TIRF microscopy, telemetry, animal models of diabetes, and isolation of intact human arteries and arterial myocytes from non-diabetic and diabetic patients.
Aim 1 will elucidate the mechanisms underlying coupled gating of Cav1.2 channels by examining the structural requirements by which CaM promotes Cav1.2 coupling.
Aim 2 will determine the functional consequences of Cav1.2 coupling in arterial myocytes and intact arteries by examining the relationship between CaM dynamics, coupled events and arterial myocyte function in intact arteries.
Aim 3 builds on the preceding aims to elucidate the importance of Cav1.2 coupling to arterial dysfunction during diabetes. We will evaluate the role of coupled events, CaM dynamics and the contributions of the signaling module orchestrated by AKAP150 in the development of vascular dysfunction during diabetes. The proposed work is innovative at the technical level, in its ability to unmask underlying mechanisms of Cav1.2 coupling, and its unique ability to integrate the results of this gating modality as it relates to C and ET coupling in a modern quantitative framework that relates to vascular complications during diabetes. Such outcomes will be significant because they will provide new fundamental information on the mechanisms by which increased coupling of Cav1.2 channels underlies vascular dysfunction during diabetes and may contribute to the development of rational therapies for the treatment of this pathological condition. Importantly, critical concepts of our model have been validated in freshly dissociated human arterial myocytes from non- diabetic and diabetic patients, thus underscoring the translational significance of our application.

Public Health Relevance

Cardiovascular complications in the USA have reached pandemic proportions fueled in part by rising obesity rates. Vascular complications associated with diabetes contribute to hypertension, heart disease and stroke. Here we will carry out careful quantitative studies to understand a new gating modality of vascular Cav1.2 channels that amplifies Ca2+ influx and regulates the function of arterial myocytes leading to vascular complications during diabetes. The results from the proposed work will identify potentially valuable therapeutic targets in the genesis and treatment of diabetic hypertension.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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OH, Youngsuk
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University of California Davis
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