The broad, long-term objectives of this proposal are to (1) characterize the structure-function relationship of the parts of sodium channels that control slow inactivation and to (2) assess the potential contribution of sodium channel slow inactivation in the mechanisms of epilepsy.
The Specific Aims are: 1) to measure the properties of sodium channels during slow inactivation and to measure the voltage sensitivity and kinetics of slow inactivation, 2) to express channels with mutations of the S4 region of repeats M and IV to determine whether that part of the sodium channel molecule controls slow inactivation, and 3) to determine the differences between the F(Vh) curves measured from normal neurons and neurons from spontaneously epileptic (tottering) mice, and to identify anti-epileptic agents that affect the voltage dependence and kinetics of slow inactivation. The health-relatedness of this proposal is its application to epilepsy; a critical factor in neuronal hyperexcitability (that could lead to epilepsy) is a right-shift in the midpoint of the F(Vh) curve which leads to an increase in the number of channels available for activation from resting potential. The kinetics, effective valence, and voltage dependence of the F(Vh) curve in crayfish giant axons will be measured using axial-wire voltage clamp. The effects of anti-epileptic drugs such as diphenyl- hydantoin, carbamasepine and sodium valporate on properties of the F(Vh) curve in crayfish will be studied. Native and mutated sodium channels will be expressed in Xenopus oocytes, and macroscopic currents will be recorded using two-electrode voltage clamp and outside-out patch configuration. Slow inactivation properties will be compared between wild-type channels and those with S4, repeats III and/or IV amino acid replacement and deletion mutations produced by site-directed mutagenesis. Properties controlling slow inactivation will be compared between neurons cultured from non-phenotypic tottering (spontaneously epileptic) mice using patch clamp techniques.
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