Voltage control of Ca²⁺ permeation through N-type calcium (Ca(V)2.2) channels

Voltage-gated calcium (Ca(V)) channels deliver Ca(2+) to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca(2+) over other cations is thought to involve multiple Ca(2+)-binding sites within the pore. Although the Ca(2+) affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca(V)2.2) channels to investigate the effect of voltage on Ca(2+) flux. We found that the EC50 for Ca(2+) permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca(2+) ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca(V)2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca(2+)-Ba(2+) anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca(2+)-Ba(2+) anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca(2+) permeation through Ca(V)2.2 channels may require at least four Ca(2+)-binding sites. Finally, our results suggest that the high affinity of Ca(2+) for the channel helps to enhance Ca(2+) influx at depolarized voltages relative to other ions (e.g., Ba(2+) or Na(+)), whereas the absence of voltage effects at negative potentials prevents Ca(2+) from becoming a channel blocker. Both effects are needed to maximize Ca(2+) influx over the voltages spanned by action potentials.