The permeation pathway of the voltage-gated Na/+ channel encompasses a selectivity filter, and an activation gate. Upon binding, local anesthetics (LAs) block this permeation pathway whereas batrachotoxin (BTX) alters ion selectivity and causes persistent Na/+ channel opening. Our long-term objectives are (1) to understand better the structure/function relationship of receptor sites for LAs and for BTX in the alpha-subunit of the voltage gated Na/+ channel and (2) to explore the interplay among the LA receptor, the BTX receptor, and the Na/+ permeation pathway. Such information will be beneficial for the design of therapeutic drugs. Two hypotheses will be tested: first, the receptors for LAs and for BTX change their conformations during state transitions and second, the LA receptor and the BTX receptor align in close proximity to each other within the Na/+ permeation pathway. Specifically, we will examine the structural basis of the static-ligand receptor interactions in the rat skeletal muscle Na/+ channel (SKM1), the successive changes in receptor sites during state transitions, and the functional roles of these receptors within the permeation pathway. Site-directed mutagenesis will be used to create mutants with point mutations at these two separate ligand binding sites. These mutants will be transiently expressed in human embryonic kidney cells. Both whole-cell and single-channel currents will be measured in order to obtain detailed kinetic information on the dynamic interactions between Na/+ channels and these ligands. A substituted- cysteine accessibility method will be used to scan the regions within or near the ligand binding sites that may change conformations during state transitions. Finally, the ion selected mutants will be examined to reveal the putative interactions between the selectivity locus and the receptors. Together, these studies should provide a better understanding of how BTX and LAs modulate the Na/+ permeation pathway at the molecular level as well as a clearer view of the dynamic interactions between these ligands and the alpha-subunit Na/+ channel.
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