The research described in this application centers on the nature of pacemaker mechanisms and their modulation in neurons. The ultimate goal is to define the set of cytoplasmic and membrane proteins that mediate this important function and to determine the nature of the modulation caused by cyclic nucleotide-dependent protein phosphorylation. In particular, monoclonal antibodies will be generated to membrane proteins in several pacemaker systems in the mollusk Aplysia. These antibodies will be screened, by immonoblots and immunocytochemistry, to select for those that uniquely recognize antigens of pacemakers systems. Such """"""""pacemaker specific"""""""" antibodies will be tested for pharmacological interference of pacemaker function by intracellular injection. Eventually, we would like to convert non-pacemaker cells into pacemaker cells by injecting the appropriate messenger RNAs (mRNAs) into non-pacemaker cells in order to determine whether all the essential constituents for pacemaker function have been isolated. This last goal may not be achieved during the first project period. For these studies, we will concentrate on three different pacemaker systems in one organism, Aplysia: 1) A transient pacemaker -- the bag cells, a group of peptidergic neurons, which release their hormonal messenger, by producing bursting pacemaker activity for approximately 30 minutes. 2) A constant pacemaker -- the identifiable neurons R15, L2, L3, L4 and L6 in the abdominal ganglion, which produce groups of impulses termed """"""""bursts"""""""", separated by a silent interburst interval lasting seconds to minutes. 3) A circadian pacemaker system -- part of the eye, which is expressed as a turning on and modulation of pacemaker activity , emitted in the optic nerve, over a 24-hour period. The following specific projects are planned in the first project period: A). Generating monoclonal antibodies to crude membrane fractions of the above three pacemaker systems. B). Screening the monoclonal antibodies for specific recognition of antigenic determinants of the three pacemaker systems by immunocytochemistry and immunoblots (Western blots). C). Attempting to interfere with pacemaker function by intracellular injection of """"""""pacemaker-specific"""""""" of the intact abdominal ganglion. D). Whole-cell voltage- and membrane patch-clamp studies of the latter two pacemaker systems in order to determine what ion channels are modulated by cyclic nucleotide-dependent protein phosphorylation.
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