Vascular smooth muscle (VSM) contractility plays an important role in determining vascular tone and arterial blood pressure. VSM contractility is regulated by a feedback mechanism whereby localized, transient increases in cytoplasmic calcium (Ca2+) activate the large conductance, Ca2+ - activated K+ channel (BK). Activation of BK causes transient membrane hyperpolarization and inhibition of Ca2+ influx. The BK betal subunit, found exclusively in smooth muscle, increases both the voltage- and Ca2+ -sensitivity of the channel formed by a tetramer of alpha subunits. The increased sensitivity is essential for an effective feedback inhibition. Decreased betal expression is associated with hypertension, and a gain-in-beta1 function is associated with a reduced incidence of diastolic hypertension, stroke and myocardial infarction. Little is known about the molecular mechanism of betal-modulation of alpha subunits. Our approach to the molecular mechanism is to identify contacts between alpha and betal and to determine the structural and functional consequences of the different contacts. In collaboration with A. Karlin, we have developed a preliminary map of the interfaces between the alpha and betal subunit, based upon the crosslinking, spontaneously and with a novel membrane-impermeant crosslinker, of cysteine residues introduced one at a time into the extracellular flanking region of each transmembrane segment of alpha arid betal. We found that the betal TM helices are in close proximity to alpha SO TM helix, the unique seventh TM helix of alpha. We propose to: (1) Determine the functional consequences of constraining the movement of one TM helix relative to another and to determine the effects of functional state on the susceptibility to crosslinking;(2) dentify and determine the structural arrangement and the functional role of the contacts in alpha betweenSO and S1-S6. Pharmacological approaches to activate BK channels represent an emerging strategy.to control membrane excitability. We identified a natural product, rottlerin, which potently activates the BK channel. We propose to determine, in collaboration with the Chemistry and Animal/Tissue culture core, whether rottlerin and its derivatives normalize vasoreactivity in hypertensive animals. The experiments proposed will uncover fundamental molecular mechanisms of betal regulation of BK alpha and may lead to a new class of direct BK-channel activators to treat hypertension and other vascular disorders.
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