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₂₁ Dihydropyridine Receptor Subunit Is a Critical Element for Excitation-Coupled Calcium Entry but Not for Formation of Tetrads in Skeletal Myotubes

Marcin P. Gach ^*, Gennady Cherednichenko , Claudia Haarmann , Jose R. Lopez ^*, Kurt G. Beam , Isaac N. Pessah , Clara Franzini-Armstrong  and Paul D. Allen ^*

^* Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts; Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California; Department of Physiology and Biophysics, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado; and Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania

Correspondence: Address reprint requests to P. D. Allen, Dept. of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7334; Fax: 617-732-6927; E-mail pdallen{at}partners.org.

It has been shown that small interfering RNA (siRNA) partial knockdown of the ₂₁ dihydropyridine receptor subunits cause a significant increase in the rate of activation of the L-type Ca²⁺ current in myotubes but have little or no effect on skeletal excitation-contraction coupling. This study used permanent siRNA knockdown of ₂₁ to address two important unaddressed questions. First, does the ₂₁ subunit contribute to the size and/or spacing of tetradic particles? Second, is the ₂₁ subunit important for excitation-coupled calcium entry? We found that the size and spacing of tetradic particles is unaffected by siRNA knockdown of ₂₁, indicating that the visible particle represents the _1s subunit. Strikingly, >97% knockdown of ₂₁ leads to a complete loss of excitation-coupled calcium entry during KCl depolarization and a more rapid decay of Ca²⁺ transients during bouts of repetitive electrical stimulation like those occurring during normal muscle activation in vivo. Thus, we conclude that the ₂₁ dihydropyridine receptor subunit is physiologically necessary for sustaining Ca²⁺ transients in response to prolonged depolarization or repeated trains of action potentials.