STRUCTURE FUNCTION AND DEVELOPMENT OF THE ACTIVE ZONE

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The long-term goals are to elucidate the mechanisms of transmitter release and differentiation o the presynaptic nerve terminal. The present proposal will focus on voltage-sensitive calcium channels (VSCCs) and the active zone (site of transmitter release) at the neuromuscular junction (NMJ). Novel synthetic omega conopeptides and dihydropyridine (DHP) will be used as probes to characterize VSCCs in relation to transmitter release at developing, regenerating and diseased NMJs. (I) To examine the ontogeny of VSCC subtypes at developing mammalian NMJs. Two hypotheses will be tested: (A) L-type VSCCs modulate transmitter release at developing. but not mature NMJs. The effect of DHP on synaptic potentials, and the possible involvement of Ca2+-gated K+ channels and/or somatostatin in L-type VSCC-modulated transmitter release will be studied. The presence of L-type VSCCs at developing nerve terminals will be confirmed with immunocytochemistry. (B) N-type VSCCS mediate transmitter release at developing, but not at mature NMJs. The hypothesis will be tested by physiological and morphological approaches with omega conopeptides. In addition, the notion that developing NMJs also use PIQ-type VSCCs to mediate transmitter release as in adult muscles will be examined. (II) To examine the change in VSCC subtypes at regenerating NMJs in adult muscles. The hypothesis that re-formation of adult NMJs following injury mimics embryonic development with respect to the switch in VSCC subtypes will be tested. (III) To test the hypothesis that Lambert-Eaton Myasthenic Syndrome (LEMS) antibodies cause a reduction of VSCCs from the motor nerve terminal. LEMS antibodies will be passively transferred to mice. The effect on the number of VSCCs will be studied with fluorescence microscopy and autoradiography. The proposed research would provide the first study on the ontogeny of VSCCs at developing and regenerating NMJs. Due to the lack of specific probes for VSCCs in the past, our knowledge of presynaptic differentiation has considerably lagged behind that of postsynaptic differentiation. Thus, the proposed research would yield new insights into the mechanisms on how the synapse works, forms and is repaired. The proposed work may also provide a better understanding of the etiology of human neuromuscular diseases.