In addition to the phosphorylation-dependent effects on ion channels, G-proteins have an additional "direct" effect on the regulation of cardiac Na+ channels. The non-phosphorylation-dependent Gas-mediated increase of peak/Na is not due to a change in activation, inactivation, recovery from inactivation or a change in single channel amplitude. These results suggest that the number of functional Na+ channels have increased in the membrane. We showed that the new channels are in caveolae (Yarbrough et al., 2002). Na+ channels within the caveolae membrane become functional when the caveolae "neck" fuses with the plasma membrane and opens to establish electrical continuity between the extracellular space and the intracaveolae compartment. This application focuses on determining the co-localization of Na+ and Ca2+ channels in caveolae and the role of Gas in the regulation of caveolae. Specifically, we propose to address the following questions: 1) Are Na+ and Ca 2+ channels sorted to the same or different caveolae? Yarbrough et al., (2002) showed that Na+ channels are found in caveolae membranes. In addition, our preliminary data show that the anti-a1C antibody recognizes a protein signal in the caveolae-rich fraction of rat ventricular myocytes. We hypothesize that Na+ and Ca2+ channels are sorted to the same caveolae. We will investigate this hypothesis using immunoprecipitation, Western blot analysis, immuno-fluorescence, immuno-electron microscopy techniques, and direct patch clamp recordings using the tip-dip method. 2) What is the functional role of the N-terminus of Gas in the regulation of caveolae? Lu et al., (1999) showed that Gas could enhance the size of Na+ current in a cAMP-independent fashion. Our data also show that in yeast two-hybrid screens and GST fusion studies, the N- and C-terminals, all of the intracellular segment loops, and interdomain loops of the rat cardiac Na+ channel, Galphas does not interact directly with the channel nor does Gby. However, a short N-terminal peptide of Galphas (a.a. 27-42) can mimic the effects of increasing the Na+ current. We hypothesize that the N-terminal of Galphas plays an important role in the regulation of caveolar opening. Using the Na+ current as our assay, we will examine the functional effects of the N-terminal of Gas using Galphas/Galphat and Galphas/Galphai chimeras and short N-terminal Galphas oligomers. We will also probe for the substrate that Galphas is interacting with to regulate the opening of caveolae. 3) Caveolae are dynamic omega-shaped structures whose membrane fusion and fission mechanisms are virtually unknown. This specific aim will test the involvement of membrane-associated proteins in caveolar docking and/or fusion events. We will also probe for the substrate that Gas is interacting with to regulate the opening and closing of caveolae necks. Caveolae in endothelial cells have been shown to contain key proteins known to mediate vesicle formation, docking, and/or fusion. We will test for the involvement of toxin-sensitive synaptobrevin (VAMP, Vesicle-Associated Membrane Protein) in cardiac ventricular caveolar docking and/or fusion using antibodies recognizing members of the VAMP family and by the neurotoxin, tetanus toxin, which proteolytically cleave VAMP proteins. We will assay changes in the Na+ current. The localization of VAMP on caveolar structures will be tested using immunohistochemical approaches with antibodies to VAMP and Caveolin-3. Co-localization of VAMP with Cav-3 will be important to identify the omega -shaped membrane structure as a caveolae rather than another kind of vesicle.