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Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103

Correspondence to Roman Shirokov: roman.shirokov{at}umdnj.edu

Using the lanthanide gadolinium (Gd³⁺) as a Ca²⁺ replacing probe, we investigated the voltage dependence of pore blockage of Ca_V1.2 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.

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