Calmodulin regulates Cav3 T-type channels at their gating brake
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Date
2017-12-08
Authors
Chemin, Jean
Taiakina, Valentina
Monteil, Arnaud
Piazza, Michael
Guan, Wendy
Stephens, Robert F.
Kitmitto, Ashraf
Pang, Zhiping P.
Dolphin, Annette C.
Perez-Reyes, Edward
Journal Title
Journal ISSN
Volume Title
Publisher
American Society for Biochemistry and Molecular Biology
Abstract
Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I–II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I–II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM.
Description
This research was originally published in the Journal of Biological Chemistry. Chemin, J., Taiakina, V., Monteil, A., Piazza, M., Guan, W., Stephens, R. F., … Spafford, J. D. Calmodulin regulates Cav3 T-type channels at their gating brake. J. Biol. Chem. 2017; 292, 20010-20031. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords
calcium channel, circular dichroism (CD), cryo-electron microscopy, gating, isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), patch clamp, short hairpin RNA (shRNA)