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J. Biol. Chem., Vol. 281, Issue 15, 10042-10048, April 14, 2006

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Functional Coupling between TRPC3 and RyR1 Regulates the Expressions of Key Triadic Proteins^*

Eun Hui Lee, Gennady Cherednichenko^¶, Isaac N. Pessah^¶, and P. D. Allen¹

From the Laboratory of Calcium Communication, Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, and ^¶Department of Molecular Biosciences and Center for Children's Environmental Health, University of California, Davis, Davis, California 95616

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 ± 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn²⁺ influx with Fura-2 and Ca²⁺ transients with Fluo-4, we found that neither excitation-coupled Ca²⁺ entry nor thapsigargin-induced store-operated Ca²⁺ entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca²⁺ entry mechanism. In Ca²⁺ imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca²⁺ release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca²⁺ release channel activity, because sarcoplasmic reticulum (SR) Ca²⁺ content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 ± 1.91-, 1.42 ± 0.08-, 2.99 ± 0.32-, and 1.91 ± 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca²⁺ release independent of SR Ca²⁺ load.

Received for publication, February 1, 2006

^* This work was supported by grants from the National Institutes of Health (P01AR17605) (to P. D. A. and I. N. P.), the Catholic University of Korea (to E. H. L.), and the Medical Research Center, Korea Science and Engineering Foundation, Republic of Korea (R13-2002-005-01002–0). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed. Tel.: 617-732-6881; Fax: 617-732-6927; E-mail: allen{at}zeus.bwh.harvard.edu.

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