Voltage-sensitive channels that mediate selective permeability to Na+ (NaV channels) are members of a family of membrane proteins that provide the physiological basis for electrical excitation in nerve, muscle and heart. The medical significance of NaV channels lies in the fact that they are the target of several classes of drugs and neurotoxins such as local anesthetics, antiarrhythmics, tetrodotoxin and saxitoxin. Genetically defective NaV channels are also responsible for various human muscle and heart disorders such as hyperkalemic periodic paralysis, paramyotonia, congenita, and long QT syndrome. Research on NaV channels has considerably advance our understanding of how channel proteins are gated by voltage and how they regulate the ionic permeability of call membranes. The long term goal of this project is to elucidate the molecular basis of ion permeation and the interaction of toxins and drugs with NaV channels. In recent years, there have been major advances in understanding how NaV channels discriminate among Na+, K+, and Ca2+. A structural motif termed the 'DEKA locus' that is composed of four residues in homologous domains I-IV has been found to play a critical role in this process.
The specific aims of this project are to: 1. Analyze the function of the DEKA locus in ion discrimination and selective permeability. 2. Define the electrostatic contribution of charged residues in the outer vestibule to ionic selectivity, conductance, and external block by inorganic cations. 3. Investigate the interaction of the DEKA locus with organic and inorganic cations that block NaV channels from the internal side. To achieve these aims, native and mutant NaV channels of the rat skeletal muscle isoform will be expressed in a cultured mammalian cell line. Macroscopic and single-channel currents will be recorded by whole-cell, patch, and planar bilayer recording techniques. The relative permeability and blocking interactions of various cations will be systematically analyzed for specific mutations of the DEKA locus and the outer vestibule. These biophysical studies will allow us deduce the functional contribution of key residues that determine ionic selectivity and unitary conductance of the NaV channel pore.
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