TY - JOUR
T1 - Competitive and non-competitive regulation of calcium-dependent inactivation in CaV1.2 L-type Ca2+ channels by calmodulin and Ca2+-binding protein 1
AU - Oz, Shimrit
AU - Benmocha, Adva
AU - Sasson, Yehezkel
AU - Sachyani, Dana
AU - Almagor, Lior
AU - Lee, Amy
AU - Hirsch, Joel A.
AU - Dascal, Nathan
PY - 2013/5/3
Y1 - 2013/5/3
N2 - CaV1.2 interacts with the Ca2+ sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α 1C. Ca2+/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca2+ ions (Ca2+-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Ca v1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaMaltered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that theNTis not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.
AB - CaV1.2 interacts with the Ca2+ sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α 1C. Ca2+/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca2+ ions (Ca2+-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Ca v1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaMaltered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that theNTis not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.
UR - http://www.scopus.com/inward/record.url?scp=84877147963&partnerID=8YFLogxK
U2 - 10.1074/jbc.M113.460949
DO - 10.1074/jbc.M113.460949
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AN - SCOPUS:84877147963
SN - 0021-9258
VL - 288
SP - 12680
EP - 12691
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -