The cardiac muscarinic K+ (KACh) current plays an important role in the parasympathetic regulation of heart rate. At present, it is generally believed that the magnitude of the KACh current is determined by the degree of G protein stimulation by acetylcholine, resulting in changes in the frequency of channel opening. However, recent studies by the applicant in excised membrane patches indicate that two intracellular molecules (ATP and an unidentified cytosolic factor) can produce as much as a 10 fold change on the open probability of the KACh channels activated by G protein, due to their marked effects on the duration of the channel open state. ATP prolongs and the cytosolic factor shortens the open time duration. These findings strongly suggest that activation of the atrial KACh current by ACh under physiological conditions may also involve such effects mediated by the two cytosolic molecules. The studies proposed in this application seek to gain understanding of the roles of ATP and the unknown cytosolic factor in KACh current activation and fast desensitization, and the mechanisms involved in these process. At present, there is a controversy as to whether ATP has any effect on the activity of the KACh channel activated by G protein. Therefore, the first specific aim is to clearly define the role of ATP in KACh current activation, particularly under more physiological conditions using rapid and short (millisecond) applications of ACh. The second specific aim is to purify the atrial cytosolic factor to homogeneity and identify it. This will help to reveal the cellular mechanism of action of the cytosolic factor as well as that of ATP on the KACh channel. The third specific aim is to examine the behavior of the KACh channel (GIRK1/CIR) expressed in oocytes which lack the activity of the atrial cytosolic factor. Using this expression system, the applicant will test whether the kinetic behavior of GIRK1/CIR is consistent with the hypothesized roles of ATP and the atrial cytosolic factor in the activation and fast desensitization of the KACh current (GIRK1/CIR).
