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

P.D. Allen,Gennady Cherednichenko,Isaac N. Pessah,Eun Hui Lee

doi:10.1074/jbc.m600981200

P.D. Allen, Gennady Cherednichenko + Show 2 more

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https://doi.org/10.1074/jbc.m600981200

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Abstract

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.

Highlights

Been reported that TRPC3-mediated Ca2ϩ entry can be induced by two different mechanisms: 1) direct activation by binding of exogenous organic molecules or endogenous metabolites such as diacylglycerol analogues [7], and 2) phospholipase C-mediated activation via phospholipase C-coupled receptor
Functional Consequence of TRPC3 Knock Down—We examined cytosolic Ca2ϩ transients in response to depolarization and direct RyR1 activation in TRPC3 KD myotubes loaded with Fluo-4
TRPC3 KD myotubes had a significantly decreased gain of EC coupling, which is reflected as a decreased cytosolic Ca2ϩ transient during EC coupling (Fig. 2)

Summary

Introduction

Been reported that TRPC3-mediated Ca2ϩ entry can be induced by two different mechanisms: 1) direct activation by binding of exogenous organic molecules or endogenous metabolites such as diacylglycerol analogues [7], and 2) phospholipase C-mediated activation via phospholipase C-coupled receptor. We have defined the role of TRPC3 during EC coupling and muscle contraction in primary mouse skeletal muscle myoblasts/myotubes in which TRPC3 mRNA and protein expression was reduced (Ͼ97 and Ͼ94%, respectively) by using small interference RNA.

Results

Conclusion