The long-term goal of this research is to understand the molecular basis of synaptic transmission in the nervous system, in particular the mechanisms by which synaptic receptors are activated by their transmitters. We are studying acetylcholine receptors expressed in tissue- cultured cells. Our experimental approach is use recombinant DNA technology to snake point mutations in the receptor, to express the mutant receptors in tissue-cultured cells, and to use single-channel kinetic methods to determine how the function of the receptors has been altered by the mutation. The first objective is to understand the mechanisms by which molecular recognition is achieved at the transmitter binding site. We want to understand l) the mechanisms by which the receptor protein distinguishes transmitter molecules from chemically-similar inorganic ions, and 2) which aspects of the transmitter-receptor interaction determine both the rates and equilibria of the activation reactions. The second objective is to investigate the molecular events that constitute the opening and closing of the receptor-linked pore, that is, channel gating. We will investigate 3) which parts of the receptor determine the rates at which the channel opens and closes, and 4) the molecular mechanisms by which the binding site and pore coupled. The third objective is to study the biophysical basis of slow channel congenital myasthenic syndrome (SCCMS), a human disease that arises from natural mutations in the acetylcholine receptor protein. 5) We will study the molecular mechanisms by which these mutations make the receptor dysfunctional. The proposed research will extend our knowledge of the fundamental operation of receptors, and will increase the likelihood that we will be able to apply this knowledge to the treatment of SCCMS and other diseases of cholinergic synapses that involve nicotinic acetylcholine receptors.
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