The long term aim of this project is the understanding of the molecular events underlying the gating of voltage dependent channels. In the present proposal, experiments are designed to describe in more detail the kinetic properties of Na and K channels and to correlate the function of the channel with known changes in the primary structure. Channels will be expressed in Xenopus oocytes and the function will be probed by measuring gating currents, macroscopic ionic currents, single channel recordings, fluctuation of gating currents and membrane admittance. There are four specific aims. 1) Studies on the function of the Shaker K channel, cloned squid K channels and Na rat brain type IIA channels to complete the description of the kinetic states of the channels. 2) Correlation between structure and function. This will be approached by studying changes in the function product by neutralization of basic and acidic residues known to produce changes in the voltage dependence of the conductance. Gating charge will be determined in the same membrane where the channels will be counted using fluctuation analysis of the ionic currents or particle counting in freeze fracture micrographs. Fluctuation analysis of gating currents will be used to determine changes in the elementary charge involved in gating. 3) Dielectric properties of membrane proteins. By measuring complex capacitance of the oocyte membrane where a known amount of protein (as determined by particle counting) is expressed, the investigator will try to assess how the electric field orients dipolar structures within the protein. This study will begin with Shaker K channel in the inactivated state (holding at O mV) and membrane proteins which are not voltage gated and it will include rhodopsin whose structure is known to about 9A resolution. 4) Modeling -- this part of the proposal will put together the other three aims and will help in directing the type of experiments according to predictions of models based on the hypothesis generated by previous experiments. Initially kinetic models will be explored and as the results of structure-function correlation emerge, more physical models will be proposed. These experiments are expected to give the investigators more insights into the molecular origin of voltage dependence which is a fundamental property of many membrane proteins with basic relevance in cell homeostasis and excitability.
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