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Vol. 15, Issue 12, 5408-5419, December 2004

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Differential Contribution of Skeletal and Cardiac II-III Loop Sequences to the Assembly of Dihydropyridine-Receptor Arrays in Skeletal Muscle

Hiroaki Takekura ^*  , Cecilia Paolini ^*  , Clara Franzini-Armstrong , Gerlinde Kugler ^|| ¶, Manfred Grabner ^||, and Bernhard E. Flucher ^¶ #

Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104; ^|| Department of Medical Genetics, Molecular and Clinical Pharmacology Innsbruck Medical University, A-6020 Innsbruck, Austria; and ^¶ Department of Physiology and Medical Physics, Innsbruck Medical University, A-6020 Innsbruck, Austria

Submitted May 19, 2004; Accepted September 8, 2004
Monitoring Editor: Daniel Goodenough

The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein–protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR _1S subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (_1S-null) myotubes reconstituted with distinct ₁ subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that _1S and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, _1C and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an _1S-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-à-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.

Abbreviations used: DHPR, dihydropyridine receptor; e-c coupling, excitation-contraction coupling; GFP, green fluorescent protein; RyR1, type 1 ryanodine receptor; SR, sarcoplasmic reticulum; T-tubule, transversal tubule.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

^* These two authors contributed equally to this work.

Present address: Department of Physiological Sciences, National Institute of Fitness and Sports, Kanoya, Kagoshima, 891-2393, Japan

Present address: Ce.S.I. Center for Research on Aging, Fondazione "G.d'Annunzio," Chieti, Italy.

^# Corresponding author. E-mail address: bernhard.e.flucher{at}uibk.ac.at.

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