Calcium channels are signaling molecules of vital importance to vascular function. These membrane-spanning proteins allow the relatively selective transfer of Ca2+ ions and are found in various cells within blood vessels, including smooth muscle cells, sympathetic nerve endings, and endothelial cells. Proper communication between sympathetic neurons and smooth muscle or between endothelium and smooth muscle is important for the normal function but derangement of this communication may contribute to pathophysiology (e.g. vasospasm or hypertension). The overall aim of this project is to explore key properties of Ca2+ channels in blood vessels. We will focus on Ca2+ channels in vascular smooth muscle or endothelial cells. In project 1A, we will study structure-function relationships of cloned L- type channels typical of vascular smooth muscle. We will clarify the structural basis of some basic L-type Ca2+ channel functions: responsiveness to Ca2+ antagonists, divalent cation selectivity, and voltage-dependent gating. We will test the hypothesis that some or all of these aspects of Ca2+ channel function arise from specific domains within the alpha1 subunit. By constructing molecular chimeras between L-type channels and other Ca2+ channels, and expressing these oocytes, we will determine which domains are necessary or sufficient for particular channel functions: (1) pharmacology -- what are the molecular determinants for DHP, phenylalkylamine or benzothiazipine actions? (2) permeation -- what regions of the Ca2+ channel structure are responsible for selectivity among divalent cations such as Ca2+ or Ba2+? What are the effects of deleting the putative intracellular Ca2+ binding site? (3) gating -- which parts of the Ca2+ channel control the voltage-dependence of gating? What are the functional consequences of alternative splicing, particularly in region IVS3? Project 1B is concerned with mechanosensitive Ca2+-permeable channels in endothelial cells (MOCs). These include stretch-activated or flow sensitive channels. We will address some basic questions: (1) How does endothelial cell [Ca2+]i respond to mechanical stimuli such as shear stress or stretch? (2) Does the response require changes in membrane potential? (3) What are the basic physiological and pharmacological properties of MOCs in endothelial cells? (4) Can MOCs be pharmacologically blocked in a potent and selective manner? (5) Are MOCs important for the overall [Ca2+]i response to mechanical stimuli?
Showing the most recent 10 out of 38 publications
