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• W2761365854 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. 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. Calmodulin (CaM) 3The abbreviations used are: CaMcalmodulinaaamino acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolITCisothermal titration calorimetryCaMBCaM bindingANOVAanalysis of varianceTFEtrifluoroethanolBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidNSCaTEN-terminal spatial Ca2+-transforming elementRFPred fluorescent proteinHSQCheteronuclear single-quantum coherencedansyl5-(dimethylamino)naphthalene-1-sulfonyl chlorideCARAcomputer-aided resonance assignmentESI-MSelectrospray ionization-mass spectrometryEGFPenhanced GFP. is a universal resident calcium sensor that promotes a calcium-dependent regulation at a canonical IQ motif of the C-terminal tails of voltage-gated calcium channels (1Ben-Johny M. Dick I.E. Sang L. Limpitikul W.B. Kang P.W. Niu J. Banerjee R. Yang W. Babich J.S. Issa J.B. Lee S.R. Namkung H. Li J. Zhang M. Yang P.S. et al.Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels.Curr. Mol. Pharmacol. 2015; 8: 188-205Crossref PubMed Scopus (35) Google Scholar2Ben-Johny M. Yang P.S. Niu J. Yang W. Joshi-Mukherjee R. Yue D.T. Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.Cell. 2014; 157: 1657-1670Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Budde T. Meuth S. Pape H.C. Calcium-dependent inactivation of neuronal calcium channels.Nat. Rev. Neurosci. 2002; 3: 873-883Crossref PubMed Scopus (167) Google Scholar, 4Halling D.B. Aracena-Parks P. Hamilton S.L. Regulation of voltage-gated Ca2+ channels by calmodulin.Sci. STKE. 2006; 2006: er1PubMed Google Scholar5Minor Jr., D.L. Findeisen F. Progress in the structural understanding of voltage-gated calcium channel (CaV) function and modulation.Channels. 2010; 4: 459-474Crossref PubMed Scopus (89) Google Scholar) and sodium channels (6Sarhan M.F. Tung C.C. Van Petegem F. Ahern C.A. Crystallographic basis for calcium regulation of sodium channels.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 3558-3563Crossref PubMed Scopus (103) Google Scholar). CaM regulates a rapid and robust inactivation gating of L-type calcium channels (e.g. Cav1.2) (7de Leon M. Wang Y. Jones L. Perez-Reyes E. Wei X. Soong T.W. Snutch T.P. Yue D.T. Essential Ca2+-binding motif for Ca2+-sensitive inactivation of L-type Ca2+ channels.Science. 1995; 270: 1502-1506Crossref PubMed Scopus (241) Google Scholar) or skeletal muscle sodium channels (e.g. Nav1.4) (2Ben-Johny M. Yang P.S. Niu J. Yang W. Joshi-Mukherjee R. Yue D.T. Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.Cell. 2014; 157: 1657-1670Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and a facilitation of channel currents in synaptic (e.g. Cav2.1) calcium channels (8Lee A. Scheuer T. Catterall W.A. Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.J. Neurosci. 2000; 20: 6830-6838Crossref PubMed Google Scholar). The conservation of CaM binding extends to basal Cav1 L-type channels in single-celled eukaryotes, such as ciliate Paramecium tetraurelia, which has an extended C terminus that includes a conserved IQ motif (9Taiakina V. Boone A.N. Fux J. Senatore A. Weber-Adrian D. Guillemette J.G. Spafford J.D. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.PLoS One. 2013; 8: e61765Crossref PubMed Scopus (29) Google Scholar). CaM mutants of Paramecium (10Kink J.A. Maley M.E. Preston R.R. Ling K.Y. Wallen-Friedman M.A. Saimi Y. Kung C. Mutations in paramecium calmodulin indicate functional differences between the C-terminal and N-terminal lobes in vivo.Cell. 1990; 62: 165-174Abstract Full Text PDF PubMed Scopus (121) Google Scholar) influence the calcium-dependent inactivation of L-type calcium currents (11Brehm P. Eckert R. Calcium entry leads to inactivation of calcium channel in paramecium.Science. 1978; 202: 1203-1206Crossref PubMed Scopus (336) Google Scholar, 12Brehm P. Dunlap K. Eckert R. Calcium-dependent repolarization in paramecium.J. Physiol. 1978; 274: 639-654Crossref PubMed Scopus (36) Google Scholar) and their control of ciliary beat frequency for swimming and turning behavior (13Brehm P. Eckert R. An electrophysiological study of the regulation of ciliary beating frequency in paramecium.J. Physiol. 1978; 283: 557-568Crossref PubMed Scopus (36) Google Scholar). CaM binding at the IQ motif and its conserved regulation are consistent with the observed calcium-dependent inactivation (known as CDI) even in expressed cnidarian L-type channel homolog (14Jeziorski M.C. Greenberg R.M. Clark K.S. Anderson P.A. Cloning and functional expression of a voltage-gated calcium channel α1 subunit from jellyfish.J. Biol. Chem. 1998; 273: 22792-22799Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). All vertebrate L-type channels display a calcium-dependent inactivation that ranges from minor to very robust, from Cav1.4 to Cav1.1 to Cav1.2 and Cav1.3 channels, respectively (15Ben-Johny M. Yue D.T. Calmodulin regulation (calmodulation) of voltage-gated calcium channels.J. Gen. Physiol. 2014; 143: 679-692Crossref PubMed Scopus (141) Google Scholar). A hallmark of the calcium-dependent inactivation in L-type calcium channels is its resilience even in the presence of high-calcium buffering in 10 mm EGTA or BAPTA. This resilience in calcium sensing in the presence of high-calcium buffering is conferred by a secondary CaM-binding site referred to as NSCaTE (N-terminal spatial Ca2+-transforming element) specifically contained within the N terminus of invertebrate Cav1 channels (9Taiakina V. Boone A.N. Fux J. Senatore A. Weber-Adrian D. Guillemette J.G. Spafford J.D. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.PLoS One. 2013; 8: e61765Crossref PubMed Scopus (29) Google Scholar) and mammalian Cav1.2 and Cav1.3 channels (16Dick I.E. Tadross M.R. Liang H. Tay L.H. Yang W. Yue D.T. A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.Nature. 2008; 451: 830-834Crossref PubMed Scopus (203) Google Scholar, 17Liu Z. Vogel H.J. Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin.Front. Mol. Neurosci. 2012; 5: 38Crossref PubMed Scopus (13) Google Scholar). NSCaTE is lacking outside of L-type calcium channels, but the highly conserved C-terminal IQ motif extends to members of the sodium channel family, from Nav2 channels in the early branching single-celled eukaryotes before the split of animals and fungi, such as apusozoan, Thecamonas trahens, and Nav1 channels (18Liebeskind B.J. Hillis D.M. Zakon H.H. Evolution of sodium channels predates the origin of nervous systems in animals.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 9154-9159Crossref PubMed Scopus (103) Google Scholar), including cnidarian jellyfish, which are the extant relatives of the likely ancestors containing the first metazoan nervous systems (19Anderson P.A. Holman M.A. Greenberg R.M. Deduced amino acid sequence of a putative sodium channel from the scyphozoan jellyfish Cyanea capillata.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 7419-7423Crossref PubMed Scopus (51) Google Scholar, 20Spafford J.D. Spencer A.N. Gallin W.J. A putative voltage-gated sodium channel α subunit (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus: structural comparisons and evolutionary considerations.Biochem. Biophys. Res. Commun. 1998; 244: 772-780Crossref PubMed Scopus (25) Google Scholar). The only member of the calcium and sodium channel superfamily to lack the reported CaM-binding elements (the C-terminal IQ motif and the N-terminal NSCaTE) are Cav3 T-type channels (1Ben-Johny M. Dick I.E. Sang L. Limpitikul W.B. Kang P.W. Niu J. Banerjee R. Yang W. Babich J.S. Issa J.B. Lee S.R. Namkung H. Li J. Zhang M. Yang P.S. et al.Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels.Curr. Mol. Pharmacol. 2015; 8: 188-205Crossref PubMed Scopus (35) Google Scholar, 2Ben-Johny M. Yang P.S. Niu J. Yang W. Joshi-Mukherjee R. Yue D.T. Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.Cell. 2014; 157: 1657-1670Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). calmodulin amino acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol isothermal titration calorimetry CaM binding analysis of variance trifluoroethanol 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid N-terminal spatial Ca2+-transforming element red fluorescent protein heteronuclear single-quantum coherence 5-(dimethylamino)naphthalene-1-sulfonyl chloride computer-aided resonance assignment electrospray ionization-mass spectrometry enhanced GFP. Here, 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 Cavβ subunits regulate Cav1 and Cav2 channel complexes (22Perez-Reyes E. Characterization of the gating brake in the I–II loop of CaV3 T-type calcium channels.Channels. 2010; 4: 453-458Crossref PubMed Scopus (25) Google Scholar). This gating brake governs the low-voltage dependence of T-type channels, and its absence generates a “runaway gating” phenotype (23Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. The I–II loop controls plasma membrane expression and gating of Ca(v) 3.2 T-type Ca2+ channels: a paradigm for childhood absence epilepsy mutations.J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar24Vitko I. Chen Y. Arias J.M. Shen Y. Wu X.R. Perez-Reyes E. Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel.J. Neurosci. 2005; 25: 4844-4855Crossref PubMed Scopus (158) Google Scholar, 25Arias-Olguín I.I. Vitko I. Fortuna M. Baumgart J.P. Sokolova S. Shumilin I.A. Van Deusen A. Soriano-García M. Gomora J.C. Perez-Reyes E. Characterization of the gating brake in the I–II loop of Ca(v) 3.2 T-type Ca2+ channels.J. Biol. Chem. 2008; 283: 8136-8144Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar26Baumgart J.P. Vitko I. Bidaud I. Kondratskyi A. Lory P. Perez-Reyes E. I–II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels.PLoS One. 2008; 3: e2976Crossref PubMed Scopus (44) Google Scholar). Mutations that alter the function of the Cav3.2 gating brake have been found in human patients with absence epilepsy (27Chen Y. Lu J. Pan H. Zhang Y. Wu H. Xu K. Liu X. Jiang Y. Bao X. Yao Z. Ding K. Lo W.H. Qiang B. Chan P. Shen Y. Wu X. Association between genetic variation of CACNA1H and childhood absence epilepsy.Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (317) Google Scholar, 28Liang J. Zhang Y. Chen Y. Wang J. Pan H. Wu H. Xu K. Liu X. Jiang Y. Shen Y. Wu X. Common polymorphisms in the CACNA1H gene associated with childhood absence epilepsy in Chinese Han population.Ann. Hum. Genet. 2007; 71: 325-335Crossref PubMed Scopus (29) Google Scholar). CaM-binding sequences are variable, which can range from a striking likeness to the C-terminal IQ motif from L-type channels, as in the gating brake of the basal metazoan Trichoplax adherens, to diverse sequences that generate a nanomolar affinity for CaM binding in protostome invertebrates such as pond snail LCav3 and all human (Cav3.1, Cav3.2, and Cav3.3) channel isoforms. We demonstrate that CaM facilitates the formation of α-helices in gating brake sequences, can pre-associate with Cav3 T-type channels without calcium ions, and its binding involves structural conformational changes in both N- and C-terminal pairs of EF hands in CaM and the gating brake. Dialysis of CaM binding (CaMB) peptides, or co-expression of apo-CaM (CaM1234) generates a significant hyper-polarizing shift in voltage sensitivities and faster gating kinetics, consistent with the mutant phenotype of Cav3 T-type channels lacking a gating brake in the I–II linker. Cav3 T-type channels contribute to pacemaker rhythms, such as the conducting system of the heart (29Mesirca P. Torrente A.G. Mangoni M.E. T-type channels in the sino-atrial and atrioventricular pacemaker mechanism.Pflugers Arch. 2014; 466: 791-799Crossref PubMed Scopus (35) Google Scholar) and low-threshold calcium potentials (also known as low-threshold spikes), that trigger rhythmic burst firing classically associated with thalamic neurons during non-rapid eye movement sleep and to the spike wave discharge during absence seizures (30Kim D. Song I. Keum S. Lee T. Jeong M.J. Kim S.S. McEnery M.W. Shin H.S. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking α(1G) T-type Ca2+ channels.Neuron. 2001; 31: 35-45Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 31Cheong E. Shin H.S. T-type Ca2+ channels in normal and abnormal brain functions.Physiol. Rev. 2013; 93: 961-992Crossref PubMed Scopus (102) Google Scholar). There is also significant evidence for T-type currents participating in “low threshold” neurotransmitter release (32Carbone E. Calorio C. Vandael D.H. T-type channel-mediated neurotransmitter release.Pflugers Arch. 2014; 466: 677-687Crossref PubMed Scopus (38) Google Scholar) and in the maintenance of vascular tone (33Kuo I.Y. Howitt L. Sandow S.L. McFarlane A. Hansen P.B. Hill C.E. Role of T-type channels in vasomotor function: team player or chameleon?.Pflugers Arch. 2014; 466: 767-779Crossref PubMed Scopus (27) Google Scholar). Cav3 T-type channels possess a significant “window current” that provides a small but continuous stream of calcium influx through a population of Cav3 T-type channels open at rest (34Dreyfus F.M. Tscherter A. Errington A.C. Renger J.J. Shin H.S. Uebele V.N. Crunelli V. Lambert R.C. Leresche N. Selective T-type calcium channel block in thalamic neurons reveals channel redundancy and physiological impact of I(T)window.J. Neurosci. 2010; 30: 99-109Crossref PubMed Scopus (155) Google Scholar). This calcium, available through Cav3 T-type channels at rest, is modeled to contribute to cellular proliferation during organ development, to the aberrant proliferation in many cancers (35Gray L.S. Schiff D. Macdonald T.L. A model for the regulation of T-type Ca2+ channels in proliferation: roles in stem cells and cancer.Expert Rev. Anticancer Ther. 2013; 13: 589-595Crossref PubMed Scopus (12) Google Scholar), and to the hypertrophied condition of the mammalian heart (36Cribbs L. T-type calcium channel expression and function in the diseased heart.Channels. 2010; 4: 447-452Crossref PubMed Scopus (55) Google Scholar). Classically, Cav3 T-type channels are mostly inactivated at rest, and their participation rate is steeply voltage-dependent (37Senatore A. Guan W. Spafford J.D. Cav3 T-type channels: regulators for gating, membrane expression, and cation selectivity.Pflugers Arch. 2014; 466: 645-660Crossref PubMed Scopus (24) Google Scholar). CaM’s regulation of the voltage sensitivities of Cav3 T-type channels at the gating brake thus has dramatic consequences to the participation of Cav3 T-type channels in normal functions, as well as during development and disease. Full-length mammalian Cav3.1 channels were purified from Sf9 insect cells (38Walsh C.P. Davies A. Butcher A.J. Dolphin A.C. Kitmitto A. Three-dimensional structure of CaV3.1: comparison with the cardiac L-type voltage-gated calcium channel monomer architecture.J. Biol. Chem. 2009; 284: 22310-22321Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and co-incubated with biotin–CaM complexed to a streptavidin–gold conjugate. Fig. 1A (left panel) shows a field of purified Cav3.1 (protein appears white) coupled to an electron dense (black) gold particle, indicating CaM is bound. Individual images illustrating different orientations of the single particles of Cav3.1 channels alone or calmodulin–gold alone and Cav3.1 channels in complex with calmodulin–gold are shown in montages of viewpoints on top, bottom, and middle panels, respectively, in Fig. 1A. The orientation of the channel in some images allows identification of the C terminus, a protruding, finger-like projection from the transmembrane domain of Cav3.1 as highlighted by an asterisk; a feature previously identified through single-particle electron microscopy 3-D reconstruction of Cav3.1 (38Walsh C.P. Davies A. Butcher A.J. Dolphin A.C. Kitmitto A. Three-dimensional structure of CaV3.1: comparison with the cardiac L-type voltage-gated calcium channel monomer architecture.J. Biol. Chem. 2009; 284: 22310-22321Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We confirmed the complexing of calmodulin with Cav3 T-type channels as co-immunoprecipitants in Western blottings (see Fig. 1B). CaM–GFP fusion proteins transfected and purified from HEK-293T cells and isolated on anti-GFP Sepharose beads were identified in the complex with HA-tagged Cav3.2 channels in Western blottings, labeled with anti-HA antibody, as an ∼259-kDa band (Fig. 1B, middle lane) in the presence of calcium ions (33.3 μm CaCl2). The co-immunoprecipitation of CaM–Cav3.2 channels failed in the absence of co-expressed HA-tagged Cav3.2 channels (Fig. 1B, left lane) or in the absence of CaM (Fig. 1B, right lane). Cav3 T-type channels lack the C-terminal IQ motif shared among other calcium channels (Cav1 and Cav2) and sodium (Nav1 and Nav2) channels (1Ben-Johny M. Dick I.E. Sang L. Limpitikul W.B. Kang P.W. Niu J. Banerjee R. Yang W. Babich J.S. Issa J.B. Lee S.R. Namkung H. Li J. Zhang M. Yang P.S. et al.Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels.Curr. Mol. Pharmacol. 2015; 8: 188-205Crossref PubMed Scopus (35) Google Scholar, 2Ben-Johny M. Yang P.S. Niu J. Yang W. Joshi-Mukherjee R. Yue D.T. Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.Cell. 2014; 157: 1657-1670Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Because both EM and co-immunoprecipitation experiments indicated that CaM binds to full-length mammalian Cav3.1 and Cav3.2 channels, we therefore next sought to delineate the CaM-binding domain. A unique but ubiquitous feature in Cav3 T-type channels is a helix–loop–helix, gating brake motif (similar in structure to a region in fumarase enzyme) in the proximal I–II linker that is in the analogous position of β subunit binding to Cav1 and Cav2 calcium channels (25Arias-Olguín I.I. Vitko I. Fortuna M. Baumgart J.P. Sokolova S. Shumilin I.A. Van Deusen A. Soriano-García M. Gomora J.C. Perez-Reyes E. Characterization of the gating brake in the I–II loop of Ca(v) 3.2 T-type Ca2+ channels.J. Biol. Chem. 2008; 283: 8136-8144Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). It is within the second helix of the gating brake of the I–II cytoplasmic linker where we have found an analogous region to the C-terminal IQ motif, with nanomolar affinity binding to CaM (Fig. 2A). Sequence alignments of Cav3 T-type channels (Fig. 2B) indicated a predicted CaM-binding site (illustrated by red color gradient) in helix-2 of the gating brake of representative metazoan species from cnidarians to the three human Cav3.1, Cav3.2, and Cav3.3 channels. A CaM-binding site is also predicted in Cav3 T-type channels by the on-line tool, CaM Target Database (39Yap K.L. Kim J. Truong K. Sherman M. Yuan T. Ikura M. Calmodulin target database.J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref PubMed Scopus (462) Google Scholar). Predictions suggest that the gating brake sequence is cytoplasmic, and a helical wheel analysis indicates its amphipathic nature. The most basal multicellular organism with a Cav3 T-type channel is T. adherens (a placozoan) (40Smith C.L. Abdallah S. Wong Y.Y. Le P. Harracksingh A.N. Artinian L. Tamvacakis A.N. Rehder V. Reese T.S. Senatore A. Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue.J. Gen. Physiol. 2017; 149: 483-510Crossref PubMed Scopus (24) Google Scholar), which has a region spanning the gating brake that is unlike other gating brake sequences (Fig. 2C, blue bar) with a core 17 amino acids that resembles the C-terminal CaM-binding IQ motif of Cav1, Cav2, and Nav2 channels (Fig. 2, C and D, blue bars). The Cav3 T-type channel in the basal multicellular placozoan may represent a structural intermediate involving the positioning of the C-terminal IQ motif in the common ancestor to calcium channels and sodium channels to the proximal I–II linker, before a divergence and appearance of a gating brake helix–loop–helix motif shared among all other metazoan Cav3 T-type channels. Comparatively, the IQ motif of Cav1 L-type channels is mostly identical from microbial eukaryotes to human homologs (9Taiakina V. Boone A.N. Fux J. Senatore A. Weber-Adrian D. Guillemette J.G. Spafford J.D. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.PLoS One. 2013; 8: e61765Crossref PubMed Scopus (29) Google Scholar), where the CaM-binding gating brake motif in Cav3 T-type channels is more divergent. The protein similarity of gating brake sequences between protostome invertebrate (e.g. pond snail LCav3) and among the human Cav3 isoforms is 71–76% (Fig. 2C, red bars). Differences in gating brake sequences may reflect a local adaptability of the gating brake among Cav3 T-type channels in different animals. 26-mer peptide sequences spanning the gating brake region were synthesized for snail LCav3 and human Cav3.1, Cav3.2, and Cav3.3 channels dubbed “Cav3 CaMB” peptides. In addition, a PGPGPG substituted Cav3.2 channel peptide (Cav3.2mut) was synthesized, which served as a negative control peptide in experiments (see yellow highlighted sequence in Fig. 2B). CaMB peptides promoted a gel-mobility shift with CaM in the presence of 0.1 mm CaCl2-containing solution and increasing molar ratios (0.5 to 4×) of snail LCav3 or human Cav3.1, Cav3.2, and Cav3.3 channel CaMB peptides (Fig. 3). Mutated Cav3.2 CaMB peptide (Cav3.2mut) with the PGPGPG substitution did not promote a gel-mobility shift with CaM (Fig. 3). Cav3.3 peptide has the weakest apparent interaction compared with other Cav3.1 and Cav3.2 peptides, which correlates with its weaker predicted binding affinity (Fig. 2B). Presence of urea attenuated the weakest interacting human Cav3.3 channels with CaM without affecting the higher affinity LCav3 gating brake peptide with CaM. Differential circular dichroism (CD) spectroscopy of snail LCav3, human Cav3.2, and Cav3.3 gating brake peptides suggest that the peptides assume a more helical secondary structure upon co-incubation with CaM or with helix-stabilizing agent trifluoroethanol (TFE) (Fig. 4A). Helical propensity of peptides increases after titrating higher TFE concentrations from 10 to 50% (41Jasanoff A. Fersht A.R. Quantitative determination of helical propensities from trifluoroethanol titration curves.Biochemistry. 1994; 33: 2129-2135Crossref PubMed Scopus (358) Google Scholar, 42Sönnichsen F.D. Van Eyk J.E. Hodges R.S. Sykes B.D. Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide.Biochemistry. 1992; 31: 8790-8798Crossref PubMed Scopus (617) Google Scholar). The apparent α-helical formation of LCav3 and Cav3.2 resembles the CD spectral signature with 10% TFE, whereas Cav3.3 resembles more the spectral signature with 25% TFE. Human Cav3.1 is unique among the gating brake peptides in appearing to assume an α-helical conformation when free in solution (without CaM or TFE) (Fig. 4A), and this can be explained by its high-native alanine content (Fig. 4B). Although the circular dichroism results are suggestive of induction of helical secondary structure in the peptide upon binding CaM, some of the observed ellipticity changes may be induced by tertiary structural changes, not secondary structural ones (43Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C.Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). Mutated peptide (Cav3.2mut) has no α-helical propensity as predicted due to its proline substitutions (Fig. 4, A and B). CaM binds Cav3 CaMB peptides in 0.5 mm CaCl2 solution with a 1:1 stoichiometry, at nanomolar affinities that vary from 12, 43, 187, and 383 nm for snail LCav3, Cav3.1, Cav3.2, and Cav3.3 CaMB peptides, respectively (representative data in Fig. 5 and table of parameters in Fig. 6A). The rank order of measured binding affinities determined in the isothermal titration calorimetry (ITC) (Fig. 6A) is consistent with the rank order of concentration of CaMB peptide to CaM ratio required for a saturating gel-mobility shift, 1.0, 1.5, 1.5, and >4.0× for snail LCav3, Cav3.1, Cav3.2, and Cav3.3 CaMB peptides, respectively (Fig. 3). The Cav3.3 CaMB peptide possessed the weakest CaM-binding site of all isoforms as evidenced by the lowest measured affinity with ITC and incomplete gel shift of CaM in native PAGE (Fig. 3). Besides their variable sequence and binding affinity differences for CaM, the Cav3 CaMB peptides vary in their thermodynamic properties during their association with CaM. The association appears to be entropy-driven for LCav3, LCav3.1, and Cav3.3 peptides and enthalpy-driven for Cav3.2 peptide, as illustrated by the latter’s negative enthalpy (exothermic) (Figure 5, Figure 6A).Figure 6ITC and NMR analyses indicate high-affinity, nanomolar binding, and associated conformation changes, respectively, upon Cav3 T-type channel CaM-binding peptides to N- and C-terminal lobes of CaM. A, tabulated data of ITC analysis indicates a 1:1 stoichiometry of CaM binding with a 10, 36, 196, and 383 nanomolar affinity for" @default.
• W2761365854 created "2017-10-20" @default.
• W2761365854 creator A5002485249 @default.
• W2761365854 creator A5009025335 @default.
• W2761365854 creator A5009914921 @default.
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• W2761365854 creator A5071518518 @default.
• W2761365854 date "2017-12-01" @default.
• W2761365854 modified "2023-09-27" @default.
• W2761365854 title "Calmodulin regulates Cav3 T-type channels at their gating brake" @default.
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