Murine primary afferent neurons have mu- and delta- and kappa-opioid receptors on their somatic surfaces. mu- and delta-opioid receptors are coupled to a voltage- and/or calcium-dependent potassium channels while kappa-opioid receptors are coupled to a voltage-dependent calcium channel. The identity of the potassium channels coupled to opioid receptors on prima y afferent neurons is uncertain,m but the kappa-opioid receptor is coupled to the N-type calcium channel. Opioid receptors have been shown to be coupled to the inhibitory GTP-binding regulatory protein, Gi, resulting in inhibiti n of adenylate cyclase, and thus, to a reduction of intracellular cAMP and protein kinase A activity. The N calcium current has been demonstrated to be reduced by application of forskolin and phorbol esters, compounds which lead to increased activity of protein kinase A and protein kinase C. These results suggest that kappa-opioid receptors are coupled in calcium current and that N calcium channels are subject to regulation by several forms of protein kinases. In the present application, we will investigate the coupling of opioid receptors to their ion channels. First, we propose to determine whether or not coupling of mu-, delta- and kappa-opioid receptors to their ion channel is via a pertussis toxin sensitive G protein of the Go or G1 type. Second, we will determine whether or not nonhydrolyzable GTP analogs result in reduction of N calcium current, presumably by irreversibly releasing a G protein alpha-subunit. Third, we will determine whether activated alpha o, alpha i1 or alpha i2 G protein alpha-subunits can directly reduce N calcium current. Four, we will determine whether or not the catalytic subunit of protein kinase A will reduce the N calcium current, and if so, which of the two types of catalytic subunit is effective. Five, we will determine wheth r protein kinase C regulates the N calcium current, and if so, which of the various channels. Seven, we will determine whether or not the gating properties of N calcium channels are influenced by dynorphin A, GTP analogs G protein alpha-subunits, protein kinase A and protein kinase C. These experiments will lead, in general, to a better understanding of the coupling of G protein coupled receptors to ion channels and will elucidate the regulatory roles of receptor phosphorylation produced by protein kinase A and protein kinase C. It will lead also to a better understanding of the regulatory functions of G proteins and of protein kinase A and C.
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