Skeletal muscle membranes hyperpolarize in response to beta-adrenergic (beta-Ad) stimulation. This is thought to result from activation of the membrane-bound, electrogenic Na/K-ATPase (Na pump). Beta-Ad stimulation causes a rise in cytoplasmic CAMP and a flux of Na and K across the sarcolemma; however, the mechanism of signal transduction between the beta-Ad receptor and the Na pump remains unknown.
The aims of this project are: 1) to test the hypothesis that beta-Ad membrane hyperpolarization in a tonic muscle is mediated by cytoplasmic Na extrusion, 2) to determine whether hyperpolarization is associated with phosphorylation of the Na pump, and 3) to understand the structure/function relationship between the Na pump isoforms in skeletal muscle and their regulation by the Beta-Ad receptor. Electron probe X-ray microanalysis will be used to measure, directly, concentrations of Na and K In the cytoplasm of control and adrenergically-stimulated muscles. Since Na pump activity is also regulated by cytoplasmic [Na], permeabilized muscle will be used to test whether beta-Ad activation of Na pump results from a primary increase in cytoplasmic [Na].
The second aim will be approached using protein mapping techniques and autoradiography in whole muscles with mitochondrial and myosin ATPase activities inhibited. Na pump alpha-subunits from control and beta-Ad stimulated preparations ([gamma-32P] ATP present) will be isolated and subjected to proteolytic digestion and electrophoretic separation; the presence of phosphorylation will be determined by autoradiography. Then we plan to locate the molecular domain where activation of the Na pump occurs. Phosphorylated Na pump peptide fragments will be sequenced and compared to deduced primary sequences (based on known cDNA sequences for the different isoforms of the Na pump alpha-subunit) to localize site(s) of radioactive phosphorylation incorporation. Should phosphorylation of the Na pump a-subunit not occur in beta-Ad hyperpolarization, another signal will be sought. Recombinant DNA techniques will be used to address Aim 3. Wild type or mutated cDNAs of Na pump will be expressed in cells together with a beta-Ad receptor cDNA to elucidate the mechanism of coupling between the two molecules. Mutations will be directed toward those alpha-subunit sequences found to incorporate radiolabled phosphate, and selected for altered signal transduction: beta-Ad stimulation uncoupled from membrane hyperpolarization. Three expression systems will be established, each expressing different alpha-subunit isoform (alpha-I, alpha-II or alpha-III) together with Beta-Ad receptors, to test whether the three alpha-subunit isoforms are regulated similarly by beta-Ad stimulation. These experiments will provide general insights into the regulation of the Na pump and, potentially, other ion pumps.
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