Apocalmodulin and Ca2+Calmodulin-Binding Sites on the CaV1.2 Channel

Abstract

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

“…The use of membrane-attached cytosolic parts of a transmembrane protein for FRET two-hybrid testing ensures proper orientation of NT and CT relative to the plasma membrane and limits the possibility of artifactual interactions with cytosolic proteins such as CaM and CaBP1. The FRET data suggest that under low-Ca 2ϩ conditions of the resting cell, both CaM and CaBP1 interact with the pCT, corroborating the in vitro studies (26,29,30,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)45). In contrast, the NT interacts only with CaBP1 but not CaM under low-Ca 2ϩ conditions (Fig.…”

“…Both Ca 2ϩ -free (apoCaM) and * This work was supported by the Fields Center for Cardiovascular Research Ca 2ϩ -bound CaM (Ca 2ϩ /CaM) bind the ␣ 1C subunit of Ca V 1.2. CDI occurs when apoCaM, presumably anchored at ␣ 1C , binds Ca 2ϩ that enters the cell through the open channel and promotes a fast inactivation process via a conformational change of a yet unclear nature in Ca V 1.2 (23)(24)(25)(26)(27)(28)(29)(30)(31). A structural element in ␣ 1C that is essential for CDI is the proximal CT (pCT), which contains three CaM binding structural elements: two in the pre-IQ and a high affinity site under elevated Ca 2ϩ conditions in the IQ domain (see Fig.…”

“…A structural element in ␣ 1C that is essential for CDI is the proximal CT (pCT), which contains three CaM binding structural elements: two in the pre-IQ and a high affinity site under elevated Ca 2ϩ conditions in the IQ domain (see Fig. 1A) (26,29,30,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41).…”

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

“…tethered (66 -69) to the C terminus of the channel in a region that comprises the consensus isoleucine-glutamine (IQ) motif (67,70) as well as adjacent domains (71)(72)(73)(74)(75). Although controlled by complementary molecular determinants, VDI and CDI gating are strongly coupled (52), such that a change in the stability of the open state is predicted to affect both VDI and CDI gating (64).…”

Binding of trifluoperazine to apocalmodulin revealed by a combination of small-angle X-ray scattering and nuclear magnetic resonance

“…This was confirmed by crystallographic and nuclear magnetic resonance (NMR) studies on various target Ca 2+ CaM-binding proteins including MLCK, CaMdependent protein kinase (CaMK) and myristoylated alanine-rich Ckinase substrate (MARCKS) (reviewed in Ikura et al, 2002;Yamniuk & Vogel, 2004;Ikura & Ames, 2006). In contrast, although the interactions of apoCaM have been studied with some target proteins such as neurogranin, cyclic nucleotide phosphodiesterase (PDE), small conductance Ca 2+ -activated K + channel (SK channel), voltagegated sodium channel, MLCK and myosin V (Cui et al, 2003;Yuan et al, 1999;Mori et al, 2003;Tsvetkov et al, 1999;Hill et al, 2000;Bayley et al, 2003;Akyol et al, 2004;Tang et al, 2003;Jin et al, 2005;Houdusse et al, 2006), structural studies are very rare (Izumi et al, 2001;Schumacher et al, 2004).…”