We continue work on two projects: the first is on sodium channel gating and the second is on channel structure-function relationships, and more specifically on selectivity. The goal of the gating project is to elucidate the molecular mechanism of sodium channel function. We record currents from single sodium channels in neuroblastoma and other cultered cells, and use several procedures to define the occupancy, as a function of time, of various operationally defined conformational states of the channel protein. These states are: (1) the open state (conducts current), (2) the inactivated stated (is an absorbing state at depolarized voltages), (3) the adjacent-to-open state (occupancy is proportional to the rate of entry into the open state) and (4) the residual state (not one of first three). This system is treated as a Markov or semi-Markov process, and we have developed methods to estimate the transition rates between the various states. These rate constants, determined for a range of membrane potentials, form the basis for developing a theory for the molecular mechanisms underlying gating. The goal of the second project is to relate channel structure to function. The strategy is as follows: (1) obtain cDNA clones coding for the channel to be studied and modify the DNA sequence so that a different amino acid is specified in a particular location by using site directed mutagenesis; (2) make mRNA coding for the altered channel protein in vitro; (3) inject the mRNA into an appropriate cell type to obtain expression of the altered channel; (4) study modifications of channel function in this expression system and related altered structure and function through an appropriate theoretical analysis. We will start with an analysis of acetylcholine receptor channel selectivity.
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