Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter

Using the lanthanide gadolinium (Gd3+) as a Ca2+ replacing probe, we investigated the voltage dependence of pore blockage of Ca(V)1.2 channels. Gd+3 reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd3+ at concentrations used (< 1 mu M) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose-response curves for the two blocking effects of Gd3+ shifted in parallel for Ba2+, Sr2+, and Ca2+ currents through the wild-type channel, and for Ca2+ currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd3+. The correlation indicates that Gd3+ binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the pre-pulse voltage in the range -60 to -45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (-100 to -45 mV) and of use-dependent block (-40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd3+ decreases with concentration of Ba2+, indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd3+ binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.