title: C terminus L-type Ca2+ channel calmodulin-binding domains are 'auto-agonist' ligands in rabbit ventricular myocytes.
abstract: L-type Ca2+ channel C terminus calmodulin (CaM)-binding domains are molecular determinants for Ca(2+)-CaM-dependent increases in L-type Ca2+ current (ICa), and a CaM-binding IQ domain mimetic peptide (IQmp) increases L-type Ca2+ channel current by promoting a gating mode with prolonged openings (mode 2), suggesting the intriguing possibility that CaM-binding domains are 'auto-agonist' signalling molecules. In order to test the breadth of this concept, we studied the effect of a second C terminus CaM-binding domain (CB) mp (CBmp), in conjunction with IQmp, on single L-type Ca2+ channel currents in excised cell membrane patches from rabbit ventricular myocytes. Here we show that both CBmp and IQmp are agonist ligands that non-additively increase L-type Ca2+ channel opening probability (Po) by inducing mode 2 gating. CBmp and IQmp agonist effects were lost under conditions favouring calcification of CaM (Ca(2+)-CaM, 150 nM free Ca2+ and 10-20 microM CaM), but persisted in the presence of CaM (0-20 microM) under conditions adverse to Ca(2+)-CaM (20 mM BAPTA), indicating that CaM-binding domains increase L-type Ca2+ channel Po by a low Ca(2+)-CaM activity mechanism. Increasing Ca(2+)-CaM in the bath (cytosol) reduced the efficacy of CBmp and IQmp signals with Ba2+ as charge carrier, suggesting that CaM binding motifs target a site outside of the pore region. We measured the combined effects of CBmp and Ca(2+)-CaM-dependent protein kinase II (CaMKII) on L-type Ca2+ channels by using an engineered Ca(2+)-CaM-independent form of CaMKII that remains active under low Ca(2+)-CaM conditions, permissive for CBmp signalling. CBmp and CaMKII increased L-type Ca2+ channel Po in a non-additive manner, suggesting that low and high Ca(2+)-CaM-dependent L-type Ca2+ channel facilitation pathways converge upon a common signalling mechanism.
authors.affiliation: Department of Internal Medicine, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Igor
authors.initials: I
authors.lastname: Dzhura
authors.forename: Yuejin
authors.initials: Y
authors.lastname: Wu
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Colbran
authors.forename: Susan L
authors.initials: SL
authors.lastname: Hamilton
authors.forename: Mark E
authors.initials: ME
authors.lastname: Anderson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004594:Electrophysiology; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D008024:Ligands; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D011817:Rabbits
chemical_list: D002120:Calcium Channel Agonists; D020746:Calcium Channels, L-Type; D002147:Calmodulin; D008024:Ligands; D010455:Peptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases