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.
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