Sequence Differences in the IQ Motifs of CaV1.1 and CaV1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation

Abstract

The proximal C terminus of the cardiac L-type calcium channel (Ca V 1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (Ca V 1.1). However, there are several Ca V 1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca V 1.2 residues, including three of the "IQ motif." To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of Ca V 1.1 and Ca V 1.2. We found that in the presence of Ca 2؉ (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca V 1.2 but not of Ca V 1.1. Furthermore, replacement of two residues (Tyr 1657 and Lys 1662 ) within the IQ motif of a C-terminal Ca V 1.2 construct with the divergent residues of Ca V 1.1 (His 1532 and Met 1537 ) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca V 1.1 caused a gain of CaM binding. In full-length Ca V 1.2, substitution of these same two divergent residues with those of Ca V 1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca V 1.2 and Ca V 1.1 has a profound effect on both CaM binding and calciumdependent inactivation.

Calmodulin Interactions withCav1 andCav2 Voltage‐Gated Calcium ChannelIQDomains

“…[137][138][139][140][141] Nevertheless, Ca V 1.1 subunits do have some form of CDI 142 and an IQ domain that is capable of binding Ca 2+ /CaM. 23,143 The 1.94-Å resolution structure of Ca 2+ /CaM-Ca V 1.1 IQ domain complex 23 (PDB: 2VAY) reveals a parallel binding conformation that is very similar to that observed in the Ca V 1.2 complexes (Table 2). These crystals were centered monoclinic, space group C2, a = 84.92Å, b = 34.67Å, c = 62.98Å, α = γ = 90.00°, β = 113.59°, contained one complex in the asymmetric unit, and were grown in 32% PEG 3500, 50 mM MgCl 2 , 50 mM Tris.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…CaM is also known to bind to and regulate Ca v 1.1 and Ca v 1.2 (Tang et al 2002;Ohrtman et al 2008;Halling et al 2009). CaM contains four EF-hand Ca 2þ binding pockets (two in the carboxy-terminal domain and two in the amino-terminal domain of the protein) and binds to one site per RyR subunit (four per tetramer) (Moore et al 1999a).…”

Calcium-dependent protein folding in amyotrophic lateral sclerosis

“…At higher Ca 2+ concentrations (>1 M), it inhibits all three RyR isoforms [98]. Further, calmodulin is also known to regulate Ca v 1.1 and Ca v 1.2 [99,100]. Unfortunately, there are no detailed studies of the role of calmodulin in ALS in the literature.…”