Murine Models of Presynaptic Neuromuscular Disease

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Several human neuromuscular disorders which present as skeletal muscle weakness are associated with presynaptic impairment of release of acetylcholine (ACh). These include botulism, envenomation by certain poisonous snakes and arthropods as well as several human congential myasthenic syndromes. The best characterized of the presynaptic neuromuscular disorders is Lambert-Eaton Myasthenic Syndrome (LEMS) which often associates with oat cell carcinoma. Skeletal muscle weakness in LEMS is thought to result from autoimmunic attack on the Ca channel functional complex at the motor nerve terminal. The objective of this project is to study animal models with disruptions of motor nerve terminal ACh release to learn more about the etiology of diseases such as LEMS. Multiple subtypes of Ca channels such as the N-, P/Q-, R- and L-type exist in motor nerve terminals. P/Q-type Ca channels are primarily responsible for controlling ACh release at mammalian neuromuscular junctions, and hence are the presumed primary autoimmune target in LEMS. Autoantibodies are thought to target primarily to CH subunit of the channel, although other proteins have also been postulated as antigenic targets in LEMS. In the passive transfer model of LEMS in mice, the phenotype of Ca channel controlling ACh release from motor terminals changes, and expresses a new complement of Ca channels. In this project, intracellular microelectrode recordings of synaptic potentials, perineurial recordings of nerve terminal Ca channel activity, recordings of FM1-43 fluorescence and nerve terminal Ca channel subunit immunocytochemistry will be used to study neuromuscular transmission following passive transfer of LEMS to mice in which the a1A subunit of the P/Q-type of Ca channel is genetically-altered, either by deletion or point mutation, or in which the normal (3 subunit which coexpresses with the P/Q-type Ca channel CH subunit is mutated. The aims are: 1. Can LEMS be passively transferred to genetically-altered mice lacking functional or having mutated P/Q-type Ca channels and do they respond differently than do wildtype mice? Do these mice express L-type Ca channel activity normally, and is it altered after passive transfer of LEMS? Do L-type channels play a greater role in regulatingACh release in motor nerve terminals of mice missing the a1A gene of the P/Q-type Cachannel or in which mutations such as "tottering" or "leaner" occur in the a1A subunit? Do L-type Cachannels become present or unmasked uner either of these condidtions? 2. Which Ca channels are involved in nerve-evoked release of ACh from nerve termianls of animals lacking P/Q-type Ca channels, or having mutations in the pore-forming region? 3. What role does the Ca channel /3 subunit play in ACh release at neuromuscularjunctions? Will animals having a mutation ("lethargic") in the j84 subunit be susceptible to induction of LEMS? Results of this study should provide a better understanding of the etiology of LEMS and subsequent changes that occur at motor nerve terminals in patients with LEMS.