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J. Biol. Chem., Vol. 283, Issue 43, 29301-29311, October 24, 2008

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Sequence Differences in the IQ Motifs of Ca_V1.1 and Ca_V1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation^*

Joshua Ohrtman¹², Barbara Ritter¹, Alexander Polster, Kurt G. Beam, and Symeon Papadopoulos³

From the Department of Physiology, OE 4200, Hannover Medical School, Hannover, Germany 30625 and the Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, Colorado 80045

The proximal C terminus of the cardiac L-type calcium channel (Ca_V1.2) 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_V1.1). However, there are several Ca_V1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca_V1.2 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_V1.1 and Ca_V1.2. We found that in the presence of Ca²⁺ (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca_V1.2 but not of Ca_V1.1. Furthermore, replacement of two residues (Tyr¹⁶⁵⁷ and Lys¹⁶⁶²) within the IQ motif of a C-terminal Ca_V1.2 construct with the divergent residues of Ca_V1.1 (His¹⁵³² and Met¹⁵³⁷) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca_V1.1 caused a gain of CaM binding. In full-length Ca_V1.2, substitution of these same two divergent residues with those of Ca_V1.1 (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_V1.2 and Ca_V1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.

Received for publication, July 8, 2008 , and in revised form, August 15, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant NS24444 (to K. G. B.). This work was also supported by grants from the Deutsche Forschungsgemeinschaft and Deutscher Akademischer Austauschdienst (DAAD) (to S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Partially supported by National Institutes of Health Training Grant HD41697.

³ To whom correspondence should be addressed: Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Fax: 49-511-532-2938; E-mail: papadopoulos.symeon{at}mh-hannover.de.

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