Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirusexpressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both Nand C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca 2؉

Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.

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and Ser 635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process. Endothelial nitric-oxide synthase (eNOS)1 is an important enzyme in the cardiovascular system producing nitric oxide (NO), a key regulator of blood pressure, platelet function, and vessel remodeling. Endothelial NOS is regulated by multiple mechanisms involving both protein-protein interactions with several different proteins, including caveolin-1 and Hsp90 (1), and post-translational modifications that include Nmyristoylation, cysteine palmitoylation, and multisite phosphorylation. The two most thoroughly studied phosphorylation sites have been the activation site, human Ser 1177