Gating of single N-type calcium channels recorded from bullfrog sympathetic neurons

Abstract

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the piper, mean N-channel open probability (P(o) measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., P(o) at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τ(o)) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τ(sh1) = 0.3 ms) did not vary with the test potential, while the longer shut time component (τ(sh2)) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel P(o) during individual sweeps at +40 mV was often high (~0.8), mean P(o) was reduced by null sweeps, low P(o) gating, inactivation, and slow activation. The variability in mean P(o) across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low P(o) gating (P(o) = 0.2, τ(o) = 1 ms at +40 mV) could be sustained for ~1 min in some patches. The clustering of null sweeps and sweeps with low P(o) gating is consistent with the idea that they result from different modes of N-channel gating. While P(o) of the main N-channel gating state is high, the net P(o) is reduced to a maximum value of close to 0.5 by other gating processes.