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Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels
B Z Peterson1, J S Lee, J G Mulle
1Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA.
Abstract:
L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 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 Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) 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 Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.
Insights
L-type calcium channels rapidly inactivate via calmodulin binding. This study confirms the EF-hand motif
Area of Science:
- Molecular and Cellular Physiology
- Ion Channel Function
- Calcium Signaling
Background:
- L-type calcium channels (alpha(1C)) exhibit rapid inactivation mediated by intracellular Ca(2+).
- Calmodulin (CaM) is identified as the primary Ca(2+) sensor, binding to the alpha(1C) carboxyl tail.
- The role of an EF-hand motif in Ca(2+)-dependent inactivation has been debated.
Purpose of the Study:
- To investigate the essentiality of the EF-hand motif for Ca(2+)-dependent inactivation of L-type calcium channels.
- To elucidate the specific contribution of the EF-hand motif in the inactivation process.
Main Methods:
- Site-directed mutagenesis of the EF-hand motif and adjacent regions in alpha(1C) calcium channels.
- Electrophysiological recordings to assess Ca(2+)-dependent inactivation.
- Development of a novel inactivation model incorporating CaM multivalent binding.
Main Results:
- The EF-hand motif, particularly the VVTL amino acid cluster, is critical for Ca(2+) inactivation.
- Mutations within the VVTL cluster, especially V1548Y, significantly abolish inactivation.
- Mutating Ca(2+)-coordinating residues in the EF hand had a modest effect, suggesting a non-sensor role.
Conclusions:
- Calmodulin acts as the Ca(2+) sensor for inactivation.
- The EF-hand motif likely functions in transducing Ca(2+)-CaM binding into channel inactivation.
- This model explains inactivation properties of wild-type and mutant channels.