Gating Charge Movement Is Prevented By Open State Occupancy Of N-type (CaV2.2) Calcium Channels

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L-type calcium channels show a loose coupling between channel closing and gating charge movement. There are significant gating differences between N-type and L-type channels and we wondered if some of these differences were linked to the relationship between charge movement and channel opening. This was accomplished by comparing the time constant (τ) for channel closing (τDeact) with that for Off-gating charge movement (τ QOff) over a range of voltages. Ionic currents were recorded in 5 mM Ca2+, while gating currents were recorded in 0.1 mM La3+ and 5 mM Mg2+ (La-Mg) from N-channels expressed in HEK 293 cells. τ QOff was larger than τDeact and the voltage dependence of the τ QOff was less steep than that for τDeact, which suggests that gating charge relaxation does not limit channel closing. To determine if the reverse was true, we used roscovitine, which slows N-channel closing by holding the channel in a high Po open state. We found that τ QOff was identical to τDeact in roscovitine. There was a risk that residual ionic tail current could contaminate Off-gating current, so we used an envelope protocol to measured the recovery time course of QOn (no ionic current contamination), and found the same τ as for both τ QOff and τDeact in roscovitine. This coincidence of τ QOff with τDeact suggests that transition out of the roscovitine-bound high Po open state becomes rate limiting to both QOff and channel closing. We conclude that, unlike L-channels, the high Po N-channel open state places the channel into a confirmation that locks gating charge into the activated position.