The principal goal of the proposed research is to isolate, reconstitute and characterize voltage-gated sodium channels from mammalian brain. These channels can be identified and assayed during purification by measuring the binding of radioactive ssxitoxin which specifically blocks the channels at nanomolar concentrations. Purification of saxitoxin binding sites from detergent-extracts of rat brain membranes will be carried out using standard procedures. The functional properties of toxin binding sites at each stage of purification will be determined by reconstitution of the binding sites in planar phospholipid bilayers for assay of voltage- and neurotoxin-dependent ion currents through the reconstituted channels. This method has been used to incorporate sodium channels from native neuronal membranes into planar bilayers; in both cases single channel current fluctuations and macroscopic (multichannel) currents were analyzed. This approach should minimize the possibility that important functional properties of the channels will be lost during biochemical manipulations of the binding sites. Once purified saxitoxin binding sites are obtained and reconstituted in planar bilayers, the effects of biochemical modifications including protein phosphorylation, on the polypeptide composition and on the physiological properties of the channels will be determined and compared. A second goal of this project is to use a combined electrophysiological and biochemical approach to characterize calcium-activated potassium channels from mammalian brain that have been reconsitituted in planar phospholipid bilayers. The possible roles of protein phosphorylation and of calmodulin in regulating the calcium-activated potassium channels will be evaluated. The long-range goal of this research program is to elucidate the molecular structures of ion channels in excitable membranes and to link specific structural components of the macromolecules with such functional properties of the channels as voltage-gating, ion permeation and calcium-activation.
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