Our preliminary recordings of single Na channels in frog skeletal muscle have brought to light an unsuspected complexity of Na channel kinetics--the bursting and background modes of the late Na currents. These currents are important to study further because they have major implications for established theories of Na channel kinetics. We propose to study four specific aspects of these late currents: 1. Determine the fast kinetics of the late currents at 5-10 kHz time resolution. The measurements to date have been made at 2 kHz. Our estimates of mean open and closed times are shifted due to missed openings and closings in the record. We propose several improvements in our recordings, and a combined single channel-fluctuation measurement to extend our time resolution. 2. Measure the length and frequency of the burst mode, and determine the external factors that influence them. Bursts appear to be ended during pulses by a slow inactivation process. Our preliminary work indicates that the bursting mode itself may last much longer. We propose to compare burst length to the rate of slow inactivation. We will check the influence of holding potential, and of the cytoplasmic environment on the rate of appearance and the length of bursts. We will also measure the fast kinetics during bursts and compare them to those observed after treatment of the channel with N-bromoacetamide. 3. Further check the hypothesis that background currents are due to channels returning from normal inactivation. If the background currents are due to normal channels returning from inactivation, then their kinetics can be used to check models of Na channel's early currents. However, our preliminary results have shown that the rate of appearance of the background currents is not voltage dependent, as might be expected from such a model. We will test this hypothesis by comparing background open lifetimes and rate of appearance with appropriate parameters from the early currents. 4. Use the kinetic information from bursts and background currents to predict a kinetic model of the Na channel's fast currents. Our hypotheses on the origin of the late currents make explicit predictions of the rate constants of simple models of channel kinetics. We will determine these rates, and derive the form and magnitude of the early currents. Comparison of the predicted and the observed currents will serve to check our hypotheses and to establish a reasonable model for the Na channel.
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