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.
Highlights
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
We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels associate with CaM at helix 2 of the gating brake in the I–II linker of the channels
We show that Cav3 T-type channels possess high-affinity calmodulin binding at the “gating brake,” a helix–loop– helix motif located in the proximal I–II linker of known Cav3 T-type channels in the analogous position where accessory Cav subunits regulate Cav1 and Cav2 channel complexes [22]
Summary
CaM, calmodulin; aa, amino acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ITC, isothermal titration calorimetry; CaMB, CaM binding; ANOVA, analysis of variance; TFE, trifluoroethanol; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈtetraacetic acid; NSCaTE, N-terminal spatial Ca2ϩ-transforming element; RFP, red fluorescent protein; HSQC, heteronuclear single-quantum coherence; dansyl, 5-(dimethylamino)naphthalene-1-sulfonyl chloride; CARA, computer-aided resonance assignment; ESI-MS, electrospray ionizationmass spectrometry; EGFP, enhanced GFP. We show that Cav T-type channels possess high-affinity calmodulin binding at the “gating brake,” a helix–loop– helix motif located in the proximal I–II linker of known Cav T-type channels in the analogous position where accessory Cav subunits regulate Cav and Cav channel complexes [22]. This gating brake governs the low-voltage dependence of T-type channels, and its absence generates a “runaway gating” phenotype [23,24,25,26]. CaM’s regulation of the voltage sensitivities of Cav T-type channels at the gating brake has dramatic consequences to the participation of Cav T-type channels in normal functions, as well as during development and disease
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