Lanthanide gadolinium (Gd 3+ ) blocks Ca V 1.2 channels at the selectivity filter. Here we investigated whether Gd 3+ block interferes with Ca 2+ -dependent inactivation, which requires Ca 2+ entry through the same site. Using brief pulses to 200 mV that relieve Gd 3+ block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd 3+ block of the Ca 2+ influx that enhances inactivation. However, we also found that the extent of Gd 3+ block did not change when inactivation was reduced by abolition of Ca 2+ /calmodulin interaction, showing that Gd 3+ does not block the inactivated channel. Thus, Gd 3+ block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd 3+ site or reduces its affinity, or that Ca 2+ occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca 2+ -dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun . 201:1117–11123), and that Ca 2+ -inactivated channels conduct Na + when Ca 2+ is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol . 565:709–717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca 2+ so that Ca 2+ ion blocks the pore. A minimal model, in which the inactivation “gate” is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca 2+ .

Using the lanthanide gadolinium (Gd 3+ ) as a Ca 2+ 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 Gd 3+ 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 Gd 3+ shifted in parallel for Ba 2+ , Sr 2+ , and Ca 2+ currents through the wild-type channel, and for Ca 2+ currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd 3+ . The correlation indicates that Gd 3+ 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 Gd 3+ decreases with concentration of Ba 2+ , 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, Gd 3+ 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.