The long-term objective is to specify the voltage sensor and the pore of voltage-dependent brain K+ channels and the inactivation gate of voltage-dependent brain Na+ channels.
The specific aims are to: 1) establish a reference data set for the single channel kinetics and conductance of a rat brain K+ channel drk1, which we have recently cloned, sequenced and expressed; 2) test the effects of extensive changes in the non-core regions of drkl and engineer with recombinant methods a minimum functional structure; 3) correlate structure with function by comparing the single channels currents produced by closely related isomorphic variants; 4) produce point mutations or chimeras in regions thought to be the voltage sensor or conducting pore of 3C channels or the inactivation gate of Ne channels; 5) mutate the cytoplasmic linker in rat brain Na+II to test its role in inactivation gating. In all cases the tertiary channel structures are interpreted using models based upon known primary amino acid sequences. The hypotheses are tested by injecting transcripts from the natural isoforms and the engineered mutants into Xenopus oocytes. From a comparison of the voltage-dependence of transitions to and from the closed and open states among the different proteins, the gating structure may be inferred. From a comparison of single channel conductance in bi-ionic solutions, the permeation structure may be inferred. The results and their interpretation will be extended to human brain K+ and NC channels. Knowledge of the structures responsible for voltage gating and ionic flux will provide a rational basis for therapy of disorders such as epilepsy, Parkinsonism and Alzheimers disease.
Showing the most recent 10 out of 60 publications
