Critical determinants of Ca²⁺-dependent inactivation within an EF-hand motif of L-type Ca²⁺ channels

L-type (α(1c)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca²⁺, providing negative Ca²⁺ feedback in a diverse array of biological contexts. The dominant Ca²⁺ sensor for such Ca²⁺dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca²⁺ sensor induces channel inactivation by Ca²⁺-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of α₁c. Apart from the IQ region, another crucial site for Ca²⁺ inactivation appears to be a consensus Ca²⁺-binding, EF-hand motif, located ~100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca²⁺ inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca²⁺ inactivation. Mutating these amino acids to their counterparts in non-inactivating α(1E) calcium channels (MYEM) almost completely ablates Ca²⁺ inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca²⁺-coordinating residues in the consensus EF hand reduce Ca²⁺ inactivation by only ~2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca²⁺ sensor, in which Ca²⁺ binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca²⁺ sensor for inactivation, the EF-hand motif of α(1C) may support the transduction of Ca²⁺-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca²⁺-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca²⁺ binding of CaM.