Calmodulin Is the Ca2+ Sensor for Ca2+-Dependent Inactivation of L-Type Calcium Channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.

[1]  R. Huganir,et al.  Calmodulin Mediates Calcium-Dependent Inactivation of N-Methyl-D-Aspartate Receptors , 1998, Neuron.

[2]  S. Green,et al.  cAMP-Dependent Regulation of Cardiac L-Type Ca2+ Channels Requires Membrane Targeting of PKA and Phosphorylation of Channel Subunits , 1997, Neuron.

[3]  B. Neel,et al.  Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. , 1993, Analytical biochemistry.

[4]  G. Bernatchez,et al.  Mutations in the EF-hand motif impair the inactivation of barium currents of the cardiac alpha1C channel. , 1998, Biophysical journal.

[5]  D. Maclennan,et al.  Identification of calmodulin-, Ca(2+)-, and ruthenium red-binding domains in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. , 1994, The Journal of biological chemistry.

[6]  David T. Yue,et al.  Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels , 1994, Neuron.

[7]  G. Westbrook,et al.  Interactions of Calmodulin and α-Actinin with the NR1 Subunit Modulate Ca2+-Dependent Inactivation of NMDA Receptors , 1999, The Journal of Neuroscience.

[8]  A. Gronenborn,et al.  Solution structure of a calmodulin-target peptide complex by multidimensional NMR. , 1994, Science.

[9]  S. Linse,et al.  Determinants that govern high-affinity calcium binding. , 1995, Advances in second messenger and phosphoprotein research.

[10]  E. Ríos,et al.  Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor , 1993, The Journal of general physiology.

[11]  L. Kelly,et al.  Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene , 1992, Neuron.

[12]  T. Snutch,et al.  Functional properties of a neuronal class C L-type calcium channel , 1993, Neuropharmacology.

[13]  T. Snutch,et al.  Primary structure of a calcium channel that is highly expressed in the rat cerebellum. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[14]  H. Reuter,et al.  Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the alpha1C subunit. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. T. Yue,et al.  Submicroscopic Ca2+ diffusion mediates inhibitory coupling between individual Ca2+ channels , 1992, Neuron.

[16]  L. Birnbaumer,et al.  Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel. , 1992, The Journal of biological chemistry.

[17]  T. Murphy,et al.  L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes , 1991, Neuron.

[18]  S. Martin,et al.  Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences , 1996, Protein science : a publication of the Protein Society.

[19]  K. Yau,et al.  Identification of a Domain on the β-Subunit of the Rod cGMP-gated Cation Channel That Mediates Inhibition by Calcium-Calmodulin* , 1998, The Journal of Biological Chemistry.

[20]  J. Nakai,et al.  Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain , 1993, Neuron.

[21]  L. Byerly,et al.  A Cytoskeletal Mechanism for Ca2+ Channel Metabolic Dependence and Inactivation by Intracellular Ca2+ , 1993, Neuron.

[22]  E. Ríos,et al.  Involvement of dihydropyridine receptors in excitation–contraction coupling in skeletal muscle , 1987, Nature.

[23]  G. Yellen,et al.  Gated Access to the Pore of a Voltage-Dependent K+ Channel , 1997, Neuron.

[24]  E. Marbán,et al.  Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin , 1997, The Journal of Membrane Biology.

[25]  S. Narumiya,et al.  Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel , 1989, Nature.

[26]  K. Deisseroth,et al.  Signaling from Synapse to Nucleus: Postsynaptic CREB Phosphorylation during Multiple Forms of Hippocampal Synaptic Plasticity , 1996, Neuron.

[27]  S. Vincent,et al.  Structure and functional expression of a member of the low voltage-activated calcium channel family. , 1993, Science.

[28]  R. Huganir,et al.  Inactivation of NMDA Receptors by Direct Interaction of Calmodulin with the NR1 Subunit , 1996, Cell.

[29]  M. Greenberg,et al.  Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB , 1990, Neuron.

[30]  A. Means,et al.  Molecular mechanisms of action of calmodulin. , 1988, Recent progress in hormone research.

[31]  E. Stefani,et al.  Feedback inhibition of Ca2+ channels by Ca2+ depends on a short sequence of the C terminus that does not include the Ca2+ -binding function of a motif with similarity to Ca2+ -binding domains. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[32]  T. Vorherr,et al.  Calmodulin-binding domains: just two faced or multi-faceted? , 1995, Trends in biochemical sciences.

[33]  G. Rubin,et al.  Molecular characterization of the drosophila trp locus: A putative integral membrane protein required for phototransduction , 1989, Neuron.

[34]  A. Rhoads,et al.  Sequence motifs for calmodulin recognition , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[35]  C. Kung,et al.  Ion channel regulation by calmodulin binding , 1994, FEBS letters.

[36]  A. Houdusse,et al.  Target sequence recognition by the calmodulin superfamily: implications from light chain binding to the regulatory domain of scallop myosin. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Houdusse,et al.  Structure of the regulatory domain of scallop myosin at 2 A resolution: implications for regulation. , 1996, Structure.

[38]  R. Eckert,et al.  Calcium entry leads to inactivation of calcium channel in Paramecium. , 1978, Science.

[39]  D. Blumenthal,et al.  The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin. , 1989, The Journal of biological chemistry.

[40]  T. Vorherr,et al.  The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump. , 1989, The Journal of biological chemistry.

[41]  E. Stefani,et al.  Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes. , 1994, Biophysical journal.

[42]  K. Yau,et al.  Direct modulation by Ca2+–calmodulin of cyclic nucleotide-activated channel of rat olfactory receptor neurons , 1994, Nature.

[43]  T. Ishii,et al.  Mechanism of calcium gating in small-conductance calcium-activated potassium channels , 1998, Nature.

[44]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[45]  R. Rosenberg,et al.  Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers. , 1994, Biophysical journal.

[46]  W. N. Zagotta,et al.  Interdomain interactions underlying activation of cyclic nucleotide-gated channels. , 1997, Science.

[47]  J. Putkey,et al.  Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C. , 1989, Journal of Biological Chemistry.

[48]  É. Rousseau,et al.  Calmodulin Modulation of Single Sarcoplasmic Reticulum Ca2+‐Release Channels From Cardiac and Skeletal Muscle , 1989, Circulation research.

[49]  M. Bähler,et al.  Rat myr 4 defines a novel subclass of myosin I: identification, distribution, localization, and mapping of calmodulin-binding sites with differential calcium sensitivity , 1994, The Journal of cell biology.

[50]  K. Deisseroth,et al.  Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons , 1998, Nature.

[51]  K. Yau,et al.  Calcium-Calmodulin Modulation of the Olfactory Cyclic Nucleotide-Gated Cation Channel , 1994, Science.

[52]  A J Hudspeth,et al.  Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells , 1990, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[53]  A. Houdusse,et al.  A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. , 1996, Structure.

[54]  G. Yellen,et al.  The Activation Gate of a Voltage-Gated K+ Channel Can Be Trapped in the Open State by an Intersubunit Metal Bridge , 1998, Neuron.

[55]  I. Schlichting,et al.  Structure of the regulatory domain of scallop myosin at 2.8 Ä resolution , 1994, Nature.

[56]  A. Fabiato,et al.  Calcium and cardiac excitation-contraction coupling. , 1979, Annual review of physiology.

[57]  D. T. Yue.  Quenching the Spark in the Heart , 1997, Science.

[58]  D. Brody,et al.  Preferential Closed-State Inactivation of Neuronal Calcium Channels , 1998, Neuron.

[59]  D. T. Yue,et al.  Essential Ca2+-Binding Motif for Ca2+-Sensitive Inactivation of L-Type Ca2+ Channels , 1995, Science.

[60]  A. Brown,et al.  Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels. , 1991, The Journal of biological chemistry.