Block of CaV1.2 Channels by Gd3+ Reveals Preopening Transitions in the Selectivity Filter

Abstract

Using the lanthanide gadolinium (Gd3+) as a Ca2+ replacing probe, we investigated the voltage dependence of pore blockage of CaV1.2 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 μM) 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 prepulse 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.