The overall goal of the proposed research is to improve the understanding of how selected cardiac current systems which are regulated by the extent that the involved channels are phosphorylated, can be regulated by altering the balance point between channels in a phosphorylated state and channels in a dephosphorylated state. The roles played by protein kinases and protein phosphatases in the regulatory processes will be investigated using the channel/current system(s) deemed most appropriate for the issues or questions being addressed and applying conventional patch clamp techniques. Because the feline isoproterenol- stimulated, cAMP-dependent gCl is a current system/channel that can be activated by both protein kinase A and C (PKA & PKC) mediated channel phosphorylation, emphasis will be on this system. Other channels including gto, gCa-L and gK1 also will be studied when they offer special advantages for investigating questions for which gCl is not particularly well-suited model. This proposal has four specific aims. They are to determine if: 1) acetylcholine (ACh) can prevent or antagonize the activation of the isoproterenol (ISO)-induced ICl. 2) The ACh-mediated antagonism of ISO- induced ICl occurs as a result of both an inhibition of adenylyl cyclase (AC) and an activation of protein phosphatases (PPs). 3) PPs localized to (or remote from) the sarcolemma in the immediate vicinity of the channel are responsible for at least some of the ACh-induced antagonism of the effect(s) of channel phosphorylation. 4) ACh can activate PPs to antagonize effect(s) of phorbol ester-activated protein kinase C on ion channel activity. The applicant has selected IK1, Ito, ICa-L and ICl as the models for the study because each is regulated by g-protein- linked regulatory pathways and a change in one or more of them can be expected to have an effect on the APD. Furthermore, the natures of IK1 and Ito have been found to be modified from normal in ventricular tissues and myocytes obtained from diseased hearts and animal models of myocardial hypertrophy and ischemia. However, two channels command special interest: Cl- channels because they can be activated (stimulated by both PKA- and PKC-mediated mechanisms) and gto because it is the only current system/channel thus far identified to have undergone significant change in ventricular myocytes isolated from hypertrophied and failing hearts of cats, rats and man.
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