Functional coupling between TRPC3 and RyR1 regulates the expressions of key triadic proteins.
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 Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) 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 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) 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 Ca(2+) release independent of SR Ca(2+) load.
Laboratory of Calcium Communication, Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
Eun Hui
EH
Lee
Gennady
G
Cherednichenko
Isaac N
IN
Pessah
P D
PD
Allen
D000111:Acetylcysteine / Q000378:metabolism; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D016257:Fura-2 / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D015233:Models, Statistical; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D050052:TRPC Cation Channels / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D015854:Up-Regulation; D014966:Xanthenes / Q000494:pharmacology
D000814:Aniline Compounds; C409648:Fluo 4; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019837:Ryanodine Receptor Calcium Release Channel; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D014966:Xanthenes; D002110:Caffeine; D019284:Thapsigargin; D008274:Magnesium; D002118:Calcium; D016257:Fura-2; D000111:Acetylcysteine