The ability of a nerve or muscle cell to fire an impulse is a result of the properties of a particular membrane protein, the voltage-gated sodium channel. These channels are water filled pores through the cell membrane which open transiently in response to a decrease in the membrane voltage, allowing sodium ions to flow into the cell. An important goal of modern neurobiology is to understand the molecular mechanisms by which this channel changes its conformational state in response to the membrane voltage. The elucidation of this voltage-dependent gating mechanism is important to our understanding our understanding of the basic cellular mechanisms of the nervous system and their dysfunction during pathological conditions, such as epilepsy. This proposal focuses upon the gating of sodium channels. Two approaches will be combined: the recording of currents through individual sodium channels, and the use of agents which alter the normal channel kinetics. Single-channel recording is a powerful technique, allowing one to monitor openings and closings of an individual channel under conditions where the membrane voltage is held constant. The study of single molecules yields much more information about channel kinetics than traditional voltage-clamp experiments which are performed on large populations of channels. The pharmacological modification of channel gating has been an important tool in multi-channel (macroscopic) voltage-clamp studies of sodium channel gating. A number of agents have been found that remove the ability of the channel to turn off, or inactivate, during a maintained voltage step. The increased resolution of single-channel recording allows the examination of the mechanism of action of these agents in much greater detail than before. Two of these agents, N-bromoacetamide and Chloramine-T, will be used in these studies. The following questions will be addressed: Are the effects of these drugs simply to reduce the accesible states of the channel? Are the inactivation of open channels and those that have not opened similarly affected? Do agents that work from the outside of the membrane have different effects than those that work from the inside. Experiments will be carried out on both normal and modified channels in order to construct a kinetic model which accounts for the normal behavior of the channel and the mechanisms of modification. The ultimate goal is to find the number of possible kinetic states of the channel, the rate constants for transitions between them, the voltage dependence of each of these rate constants, and their pharmacological modification.
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