The long range goals of this project are to characterize the basic mechanisms underlying activity of voltage-dependent ion channels, and the roles that ion channels play in the electrical activity of neurons.
The specific aims for the next grant period focus on T-type calcium channels and delayed rectifier potassium channels, with emphasis on channel inactivation. Most studies will deal with biophysical characterization of these channels in a heterologous expression system, human embryonic kidney (HEK 293) cells, but experiments will also be performed on channels natively expressed in neurons (mostly, acutely isolated thalamic relay neurons). There are four specific aims: (1) Kinetics of the alpha1G T-type calcium channel. The kinetics of activation and inactivation will be examined. Specific questions include the state-dependence of inactivation and recovery, the completeness of inactivation at steady-state, and the basis of cumulative inactivation during repetitive depolarization. One goal is construction of a kinetic model, which will be used in the future both as an operational model and as a basis for structure-function studies of the molecular mechanism of channel gating. (2) Kinetics of T-currents in thalamic neurons. The properties of native T-currents will be compared qualitatively and quantitatively to alpha1G, to determine whether the kinetic model developed for alpha1G in HEK 293 cells is applicable to neuronal T-current. (3) Permeation and gating of the Kv2.1 potassium channel in low K+. The basis of the slow channel closing observed with Na+ as the permeant ion will be investigated, to determine whether it reflects effects of permeant ions on channel activation, or induction of a Na+- permeable """"""""inactivated"""""""" state. It will be determined whether long depolarizations induce changes in ion selectivity, or """"""""immobilization"""""""" of gating charge. (4) Inactivation of native potassium channels. The voltage-dependence of inactivation, and cumulative inactivation during repetitive pulses, will be examined for delayed rectifiers of frog sympathetic neurons and rat thalamic neurons, to test for inactivation from closed states.
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