Single Channel Conductance Of CaV2.2 At Physiological [Ca2+]Ext

Abstract

Ca2+ that enters through voltage-gated CaV2.2 channels and binds to a calcium sensor at the transmitter release site links membrane depolarization to activation of synaptic vesicle discharge. Recent evidence supports the hypothesis that the release site calcium sensor is within the single CaV2.2 channel domain. Thus, modeling presynaptic nanophysiology requires knowledge of the channel transport rate at physiological [Ca2+]ext. However, this value has only been determined previously for the non-presynaptic CaV1.x (L type) channel with a conductance of ∼2.4 pS at [Ca2+]ext=2 mM (Church and Stanley, JP 1996). Since at [Ba2+]ext=100 mM CaV1.x has a conductance of ∼24 pS while CaV2.2 has one of ∼14 pS we predicted that at [Ca2+]ext=2 mM the latter channel would have a conductance of ∼1.2 pS.Single calcium channels were recorded using low noise, quartz electrodes from freshly isolated chick dorsal root ganglion neurons which express virtually entirely CaV2.2 current. In the presence of [Ca2+]ext=2 mM and 2 μM nifedipine, to block CaV1.x, and 0.1 mM Ni+ to block CaV3.X, together with standard Na+ and K+ channel blockers and n-methyl-D glucamine+ as the primary cation, we noted two single inward channel conductances: ∼1.4 pS and ∼2.5 pS (N=4). The larger channel was identified as CaV2.2 since it was absent in 4 of 4 patches with ω-conotoxin GIVA (2.5 μM), a specific CaV2.2 blocker, but was present in 7 out of 8 patches with 2 mM [Ca2+ext] or [Ba2+ext] whereas the small channel remained (N=3). Thus, our data indicate that at physiological [Ca2+]ext, CaV2.2 has a much higher conductance, and hence larger single channel domain, than predicted.