Abstract
Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl− concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl− concentration by extracellular Cl− depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl− efflux early on during myogenic differentiation, and a moderate intracellular Cl− concentration is necessary for myoblast fusion.
Highlights
Myoblast differentiation and fusion, critical processes in skeletal muscle development and regeneration, are coordinated by a complex network of proteins and signaling molecules [1e3]
We firstly coexpressed LRRC8A tagged with mCerulean3 (FRET donor) and LRRC8E tagged with Venus (FRET acceptor) in C2C12 cells and observed fluorescence resonance energy transfer (FRET) during hypotonic stimulation
We provide twofold evidence for channel activity of volume-regulated anion channel (VRAC) during early C2C12 myoblast differentiation
Summary
Critical processes in skeletal muscle development and regeneration, are coordinated by a complex network of proteins and signaling molecules [1e3]. This includes a tightly regulated transmembrane movement of the cations Kþ and Ca2þ [4e8]. The action of potassium channels leads to the hyperpolarization of the differentiating cell [9e11], facilitating the activation of voltage-gated Ca2þ channels [4,12]. An increase in the intracellular Ca2þ concentration ([Ca2þ]i), due to Ca2þ influx from the extracellular space [4,6,13] or transient Ca2þ release from the endoplasmic reticulum [6,8], is essential for myoblast differentiation.
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