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J. Biol. Chem., Vol. 279, Issue 6, 4721-4728, February 6, 2004

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Structural Requirements of the Dihydropyridine Receptor _1S II-III Loop for Skeletal-type Excitation-Contraction Coupling^*

Gerlinde Kugler, Regina G. Weiss, Bernhard E. Flucher¶, and Manfred Grabner||

From the Departments of Biochemical Pharmacology and ^¶Physiology, University of Innsbruck, A-6020 Innsbruck, Austria

Residues Leu⁷²⁰-Leu⁷⁶⁴ within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) _1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (_1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca²⁺ transients (orthograde coupling) and whole-cell Ca²⁺ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp⁷³⁴-Asp⁷⁴⁸) required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala⁷³⁹, Phe⁷⁴¹, Pro⁷⁴², and Asp⁷⁴⁴) turned out to be essential for orthograde and two of them (Ala⁷³⁹ and Phe⁷⁴¹) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an -helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp⁷⁴⁴-Glu⁷⁵¹ of _1S) corresponds with significantly reduced Ca²⁺ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.

Received for publication, July 14, 2003 , and in revised form, November 18, 2003.

^* This work was supported in part by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria and Austrian National Bank Grants P13831-GEN and P16098-B04 (to M. G.) and P12653-MED and P15338-B05 (to B. E. F.). 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.

Work performed as part of Ph.D. thesis.

^|| To whom correspondence should be addressed: Dept. of Biochemical Pharmacology, University of Innsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria. Tel.: 43-512-507-3164; Fax: 43-512-507-2834; E-mail: manfred.grabner{at}uibk.ac.at.

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