Ca²⁺-dependent inactivation of Ca_V1.2 channels prevents Gd³⁺ block: Does Ca²⁺ block the pore of inactivated channels?

Lanthanide gadolinium (Gd³⁺) blocks Ca_V1.2 channels at the selectivity filter. Here we investigated whether Gd³⁺ block interferes with Ca²⁺-dependent inactivation, which requires Ca ²⁺ entry through the same site. Using brief pulses to 200 mV that relieve Gd³⁺ 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 Ca²⁺ influx that enhances inactivation. However, we also found that the extent of Gd³⁺ block did not change when inactivation was reduced by abolition of Ca²⁺/calmodulin interaction, showing that Gd³⁺ does not block the inactivated channel. Thus, Gd³⁺ 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 Gd³⁺ site or reduces its affinity, or that Ca²⁺ 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 Ca²⁺-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca²⁺-inactivated channels conduct Na⁺ when Ca²⁺ 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 Ca²⁺ so that Ca²⁺ 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 Ca²⁺.