%0 Journal Article %1 Pete_2000_1906 %A Peterson, B. Z. %A Lee, J. S. %A Mulle, J. G. %A Wang, Y. %A de Leon, M. %A Yue, D. T. %D 2000 %J Biophys. J. %K 10733970 Acid Amino Binding Biological, Biophysics, Calcium Calcium, Calmodulin, Cell Channels, Consensus Data, Feedback, Gov't, Humans, L-Type, Line, Membrane Models, Molecular Motifs, Mutagenesis, P.H.S., Potentials, Protein Proteins, Recombinant Research Secondary, Sequence Sequence, Site-Directed, Sites, Structure, Support, U.S. %N 4 %P 1906--1920 %T Critical determinants of Ca$^2+$-dependent inactivation within an EF-hand motif of L-type Ca$^2+$ channels. %U http://www.biophysj.org/cgi/content/full/78/4/1906 %V 78 %X 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.

@article{Pete_2000_1906, abstract = {L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular {C}a$^{2+}$, providing negative {C}a$^{2+}$ feedback in a diverse array of biological contexts. The dominant {C}a$^{2+}$ sensor for such {C}a$^{2+}$-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This {C}a$^{2+}$ sensor induces channel inactivation by {C}a$^{2+}$-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 {C}a$^{2+}$ inactivation appears to be a consensus {C}a$^{2+}$-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 {C}a$^{2+}$ inactivation, but that a four-amino acid cluster ({VVTL}) within the F helix of the EF-hand motif is itself essential for {C}a$^{2+}$ inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels ({MYEM}) almost completely ablates {C}a$^{2+}$ 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 {C}a$^{2+}$-coordinating residues in the consensus EF hand reduce {C}a$^{2+}$ inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation {C}a$^{2+}$ sensor, in which {C}a$^{2+}$ binds according to a classic mechanism. We therefore suggest that while CaM serves as {C}a$^{2+}$ sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of {C}a$^{2+}$-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed {C}a$^{2+}$-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent {C}a$^{2+}$ binding of CaM.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Peterson, B. Z. and Lee, J. S. and Mulle, J. G. and Wang, Y. and de Leon, M. and Yue, D. T.}, biburl = {https://www.bibsonomy.org/bibtex/2925096052e1f46b3aa273cf5a521bc54/hake}, description = {The whole bibliography file I use.}, file = {Pete_2000_1906.pdf:Pete_2000_1906.pdf:PDF}, interhash = {d42f02fc7d3e2311df77516ecfc7747a}, intrahash = {925096052e1f46b3aa273cf5a521bc54}, journal = {Biophys. J.}, key = 166, keywords = {10733970 Acid Amino Binding Biological, Biophysics, Calcium Calcium, Calmodulin, Cell Channels, Consensus Data, Feedback, Gov't, Humans, L-Type, Line, Membrane Models, Molecular Motifs, Mutagenesis, P.H.S., Potentials, Protein Proteins, Recombinant Research Secondary, Sequence Sequence, Site-Directed, Sites, Structure, Support, U.S.}, month = Apr, number = 4, pages = {1906--1920}, pmid = {10733970}, timestamp = {2009-06-03T11:21:25.000+0200}, title = {Critical determinants of {C}a$^{2+}$-dependent inactivation within an EF-hand motif of L-type {C}a$^{2+}$ channels.}, url = {http://www.biophysj.org/cgi/content/full/78/4/1906}, volume = 78, year = 2000 }