The long term goals of this proposal are to gain insight on how voltage dependent ion channels function in molecular terms, and to define the different protein conformational changes associated with transmembrane voltage changes and channel gating. Ionic currents and gating currents will be measured. The gating currents are the electrical manifestation of the movement of the voltage sensor. The experiments are planned for cloned K channels. Two main preliminary findings are key aspects of this proposal: 1. the discovery of an early peak component of charge movement recorded at high bandwidth (200 kHz) and 2. a tendency of gating current rates to saturate at extreme potentials. The early component and the tendency to saturation with voltage of gating current rates are consistent with the view the channel transits a large number of states, like in a diffusion process of the charge along an energy landscape shaped with barriers and wells.
The specific aims are: 1. To investigate the early component of gating currents. ON and OFF gating currents were preceded by a fast component. 2. To investigate the mechanism underlying the tendency of gating current rates to saturate with voltage. The gating current rates tend to saturate at high potentials. A model in which the charge diffuses along an energy landscape shaped with barriers and wells will be tested. 3. To gain insight on channel function via a characterization of the gating current noise and to define the charged residues involved. The properties of gating noise will be investigated. 4. To investigate mechanism(s) of slow inactivation by a characterization of ionic and gating current properties during long depolarizations. We will test the hypothesis that there are two types of slow inactivation and 5. To obtain a global model of Shaker K channel function from ionic and gating current data. These determinations, in conjunction with gating current noise measurements, should provide well defined constraints for the prediction of the conformational changes occurring during channel gating. This research on fundamental properties of ion channel should be valuable to the design and test therapeutic drugs.
