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 calcium-dependent inactivation.
Highlights
Time course of the Ca2ϩ currents that they generate
For our analysis of binding, we used C-terminal regions of CaV1.1 and CaV1.2, which were up to ϳ300 residues in length rather than the relatively short peptides (Ͻ60 residues, typically ϳ20) used in prior studies. These extended peptides have the advantage that they encompass all of the regions (A, C, and IQ) previously implicated in CaM binding and may preserve a more native structure, a point of importance because dissimilar crystal structures were obtained for two different IQ motif-containing peptides complexed with CaM [21, 22]
CaM binding was weakened when the divergent IQ motif residues in CaV1.2 C-terminal segments were converted to those of CaV1.1, and CaM binding was gained in CaV1.1 C-terminal segments by converting the divergent residues to those of CaV1.2
Summary
CaM, calmodulin; CT, C-terminal; FRET, fluorescence resonance energy transfer; RyR1, ryanodine receptor type 1. For our analysis of binding, we used C-terminal regions of CaV1.1 and CaV1.2, which were up to ϳ300 residues in length rather than the relatively short peptides (Ͻ60 residues, typically ϳ20) used in prior studies These extended peptides have the advantage that they encompass all of the regions (A, C, and IQ) previously implicated in CaM binding and may preserve a more native structure, a point of importance because dissimilar crystal structures were obtained for two different IQ motif-containing peptides complexed with CaM [21, 22]. To overcome the limited solubility of the extended C-terminal peptides [23], we expressed and purified them as fusions to the bacterial NusA protein [24], incubated them with CaM, cleaved the link between NusA and the C-terminal peptide with TEV protease, and analyzed the resultant protein mixtures by means of non-denaturing gel electrophoresis and mass spectroscopy With this assay system, we found that Ca2ϩ-CaM binds strongly to C-terminal segments of CaV1.2 but not to the corresponding segments of CaV1.1. Because the IQ motif differences between CaV1.1 and CaV1.2 are evolutionarily conserved, they are likely to be important for the different mechanisms of calcium handling in skeletal and cardiac muscle
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