Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels.
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.
Peterson
B Z
BZ
Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA.
Lee
J S
JS
Mulle
J G
JG
Wang
Y
Y
de Leon
M
M
Yue
D T
DT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D016384:Consensus Sequence; D005246:Feedback; D006801:Humans; D008564:Membrane Potentials; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D020746:Calcium Channels, L-Type; D002147:Calmodulin; D011994:Recombinant Proteins; D002118:Calcium