Inactivation of open and closed sodium channels determined separately

RW Aldrich, CF Stevens - Cold Spring Harbor Symposia on …, 1983 - symposium.cshlp.org
Cold Spring Harbor Symposia on Quantitative Biology, 1983symposium.cshlp.org
METHODS Most of our investigations have used neuroblastoma N1E115, but we have
occasionally studied cells of a pituitary line (GH4C0 and primary cultured rat myotubes. Na+
channels seem not to differ between these preparations. The single-channel methods used
are essentially like those described by Hamill et al.(1981); most experiments have been
performed on cell-attached patches, but occasionally we have studied channels in inside-
out or outside-out patches. Temperature was maintained near 15~ for most experiments. We …
METHODS
Most of our investigations have used neuroblastoma N1E115, but we have occasionally studied cells of a pituitary line (GH4C0 and primary cultured rat myotubes. Na+ channels seem not to differ between these preparations. The single-channel methods used are essentially like those described by Hamill et al.(1981); most experiments have been performed on cell-attached patches, but occasionally we have studied channels in inside-out or outside-out patches. Temperature was maintained near 15~ for most experiments. We have usually limited our detailed analysis to patches containing no more than about four active Na+ channels. This number has been determined by counting the maximum number of channels seen to be open simultaneously in a large number of records obtained with appropriate holding potentials and depolarizations sufficient to open channels with a relatively high probability. Typically, currents were sampled once per t00# see, but longer or shorter sample intervals were used when required. The usual depolarization epoch lasted 15 msec. For each record, we measured every open duration as well as the number of channel openings. Groups of records, usually 64, were averaged together to generate an ensemble" macroscopic" current of the sort that might be measured with the traditional voltage clamp.
In experiments designed to investigate transitions from resting to inactivated states, we used a two-or three-pulse protocol: Voltage was stepped from the holding level (a value negative enough so that no channels were inactivated) to a prepulse level sufficient to send some channels tO the inactivated state, and finally to a test level (a voltage that would reveal the number of channels still available to open). For the three-pulse protocol, a brief reset level (a value sufficiently negative to restore noninactivated channels to their normal resting state, and brief enough not to permit any inactivated channels to escape from the inactivated state) was interposed between the prepulse voltage and the test level. Test pulses were usually 5 msec or longer, and reset pulses were 1-4 msec in duration. The duration of the prepulse ranged from 0 msec to 300 msec, and was changed systematically at each voltage level to map out the time course of inactivation. The fraction of channels that entered the inactivated state during the prepulse was estimated with the test pulse by:(1) the peak prob-147
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