Signal-transduction systems that use cell-surface receptors coupled to heterotrimetric guanine nucleotide binding proteins (G-proteins) are an important mechanism of cell regulation. A wide variety of neurotransmitters and hormones, and their many synthetic analogs used as therapeutic agents, use this pathway to regulate diverse cell functions. Ion channels form a major class of effector targets in this system. The long term goal of this research is to understand the molecular and cellular basis for the regulation of ion channels by G-protein coupled receptors. This application focuses on the beta-adrenergic receptor/L-type Ca2+ channel effector pathway, which has served as a prototypical model of ion channel modulation by this signal pathway.
The Specific Aims of this project are to; 1) develop a recombinant model cell system in which adrenergic modulation of the L-type Ca2+ channel can be studied with defined components in tahe absence of multiple receptor subtypes and ion channel targets that have complicated studies in native cells; 2) characterize the modulation of L-typeCa2+ channels by each beta-adrenergic receptor subtype (beta1, beta 2 and perhaps beta3) in order to test the hypothesis that beta-adrenergic regulation of the L-type Ca2+ channel is receptor subtype-specific; 3) Identify, at the molecular level, the regions and residues of the eta-adrenergic receptor which are critical for subtype- specific interactions with the Ca2+ channel. The experimental approach will be to express cloned cDNAs of human beta- adrenergic receptor subtypes together with the cardiac L-type Ca2+ channel in a mammalian cell which is normally devoid of endogenous beta-receptors and L-type Ca2+ channels. The effects of adrenergic stimulation on single-channel and macroscopic Ca2=+ currents will be studied using the patch clamp technique. Mutant and chimeric receptors and Ca2+ channels will be generated in order to localize molecular regions responsible for the observed effects. The research will advance our fundamental understanding of the mechanisms by which sympathetic transmitters and hormones modulate L-type Ca2+ channels. The development of a recombinant model cell system for these studies will aid research on other ion channels which are regulated by G- protein coupled receptors, and will be useful for the development of therapeutic agents that alter ion channel function by targeting specific receptor subtypes.
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