The long term objective is the understanding of the voltage dependence of ionic channels at the molecular level. The study will be done first by completing the description of the electrical expression of Na and K channels of the squid using electrophysiological techniques. The primary sequences of Na and K channels of the squid will be sought to have basic structural and functional information from the same preparation. In addition, correlations of the structure with the function will be done mutating specific aminoacids that are thought to be responsible for voltage sensing in the Shaker K channel. The evaluation of the mutations will be done by measuring gating currents and gating charge fluctuations from large populations of channels expressed in Xenopus oocytes and the role of charges of specific aminoacids will be assessed in term of the gating charge. The study of the structure of the channel protein will be done with circular dichroism and electron paramagnetic resonance using the purified sodium channel from the electric eel. The study with these spectroscopic techniques will be done with channels in the open and closed conformations using batrachotoxin as a chemical channel opener and with an imposed membrane potential. The electrophysiological results will be modelled using kinetic schemes and they will be correlated with physical models emerging from the structural analysis. The knowledge of the operation of voltage dependent channels at the molecular level has importance in understanding modifications produced by drugs, toxins and anaesthetics and may help in designing drugs to modify the operation of channel in pathological conditions.
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