The underlying theme of this project continues to be to understand the molecular basis of ion-channel function. It is focused on """"""""A"""""""" channels, a class of voltage dependent K channels. The system of choice is the expression of channels in Xenopus oocytes by injection of mRNA and synthetic RNA transcripts from channel's cDNAs. The central goal is to understand the molecular basis for K channel diversity. This diversity underlies a multiplicity of neuronal firing patterns and the regulation of this diversity may play an important role in neuronal integration. Using several of the electrophysiological and molecular methods available to the Xenopus expression system, several types of """"""""A"""""""" channels will be characterized. One section is focuse on """"""""A"""""""" channels from rat brain. These channels belong to the class of """"""""A"""""""" channel which operate in the subthreshold region for action potential generation and regulate firing patterns in many neurons. Available data suggests that the functional properties of these channels depend on polypeptides encoded in at least two mRNA species of distinct molecular size which are separated by sucrose-gradient sedimentation. The largest mRNA fraction (6-8 Kb) encodes structural components of the channel since it expresses transient K currents. The small mRNA fraction (2-4 Kb) may express additional subunits or a modifying factor(s). The gating kinetics of channels expressed with total, 6-8 Kb fraction alone, and 6-8 Kb plus 2-4 Kb fractions will be studied Electrophysiological methods include two microelectrode voltage-clamp, patch-clamp with patch-pippettes of large tip diameters to study macroscopic currents at high resolution, and cell-attached and outside-out small patches to study single-channel properties. The nature of the modifying polypeptide(s) encoded in the small size mRNA fraction will be determined by cloning cDNAs for this polypeptide(s) using an expression assay. The diversity of """"""""A., currents that the oocyte can express when injected with mRNAs and mRNA fractions from different sources will be studied to determine which mRNAs are responsible for the diversity. The second part of this project utilizes synthetic RNA transcripts of cDNAs for a second class of """"""""A"""""""" channels derived from the Shaker locus Drosophila. The relationship between channel function and protein structure will be studied by comparing the properties of channels expressed by transcripts from several available cDNAs as well as new constructs.
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