Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain–Ca2+/calmodulin complex

Abstract

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca V s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca 2+ / calmodulin (Ca 2+ /CaM) and a Ca V channel consensus isoleucine-glutamine (IQ) motif: calciumdependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the highresolution structure of the Ca 2+ /CaM-Ca V 1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca 2+ /CaM lobes through sets of conserved 'aromatic anchors.' Ca 2+ /N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca 2+ /N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca 2+ /N lobe aromatic anchors uncovers their role in Ca V 1.2 CDF. Together, our data suggest that Ca V subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca V s.Voltage-gated calcium channels are the ion channels that define excitable cells 1 . These channels control cellular calcium entry in response to changes in membrane potential and are pivotal in the generation of cardiac action potentials, excitation-contraction coupling, hormone and neurotransmitter release and activity-dependent transcription initiation 1,2 . Ca V s are multisubunit complexes composed of three essential channel subunits 2 , Ca V α 1 , Ca V β and Ca V α 2 δ, plus the ubiquitous intracellular calcium sensor calmodulin (CaM) 3 . An additional subunit, Ca V γ, is associated with skeletal muscle channels, but its general importance in other tissues is unsettled 4 .The Ca V α 1 subunits are single polypeptide chains of ∼1,800-2,200 residues in which the ion-conducting pore is formed from four homologous repeats that each bear six transmembrane segments 2 . There are three Ca V subfamilies, which have diverse physiological and pharmacological properties that depend largely on the Ca V α 1 -subunit: Ca V 1.x (L-type), Ca V 2.x (2.1, P/Q-type; 2.2, N-type; 2.3, R-type) and Ca V 3.x (T-type) 1 . Large interdomain intracellular loops bridge the four transmembrane repeats of the Ca V α 1 subunit and serve as docking sites for auxiliary subunits and regulatory molecules that Competing Interests Statement:The authors declare that they have no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access control channel activity and connect Ca V channels to larger macromolecular complexes and cellular signaling pathways 5,6 .Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess 1,7 . Because Ca V s are major sources of calcium influx, Ca V activity is strongly controlled by both self-regulatory and extrinsic mechanisms that tune channel action in response to electrical...

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca 2+ ·calmodulins

“…The wrap-around or engulfing mode of Ca 2ϩ ⅐CaM binding to the IQ segment of Ca V I (Fig. 1) is very similar to that reported for the binding of Ca 2ϩ ⅐CaM to the IQ helix in isolation, with Ca 2ϩ ⅐CaM binding to the IQ region in an unusual parallel fashion (7,8).…”

“…The Ca 2ϩ ⅐CaMs are bound to 2 structurally distinct regions of the Ca V 1.2 carboxyl termini, the pre-IQ (T1609 to Q1651) and IQ (K1665 to Q1685). The pre-IQ region reveals a unique coiledcoil structure of 2 antiparallel, crossed, 11 turn ␣-helices, with 2 Ca 2ϩ ⅐CaMs bridging the 2 pre-IQ regions followed by the Ca 2ϩ ⅐CaM-IQ structure, the latter of which is equivalent to published structures (7,8). In support of the dimeric crystal, we provide evidence that [ 3 H]PN200-110 labeled, digitonin solubilized Ca V 1.2 from cardiac microsomes are a mixture of monomers and dimers.…”

Interdomain Dynamics Explored by Paramagnetic NMR

“…55 Here, we investigate the calmodulin complex with the IQ-recognition motif from the voltage-gated calcium channel Ca v 1.2 (IQ), which adopts three conformations in the crystal. 59 Using paramagnetic data from six different lanthanides generated with the previously developed CaMN60D mutant, 60 we studied the conformational space of this complex in solution using an ensemble-based approach. Our results confirm the existence of the three crystal conformations in solution and also show that four additional conformations have to be included into the ensemble to fulfill all the paramagnetic data and cross-validate optimally against unused paramagnetic data.…”

Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features

“…8). Two exceptions are Ca 2ϩ -CaM bound to a target peptide from Ca 2ϩ -CaMdependent kinase (21), and Ca 2ϩ -CaM bound to the IQ motif of the cardiac Cav1.2 calcium channel (22,23), both of which show the same polarity for the orientation of the C-lobe as that found in the apo-CaM/IQ motif (SI Fig. 8).…”