The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation–contraction) coupling. However, it has not been possible to demonstrate direct protein–protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1–RyR1 and JP2–TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca²⁺ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca²⁺ level is mainly determined by Ca²⁺ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca²⁺ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca²⁺ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca²⁺ entryway into skeletal muscle. Herein, recent studies on extracellular Ca²⁺ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca²⁺ entry and their influences on skeletal muscle function and disease.

JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca²⁺ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca²⁺ concentration. Single myotube Ca²⁺ imaging experiments showed reductions in both the excitation–contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca²⁺ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser¹⁶⁵ and in binding of JP2-S165F to the Ca²⁺ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca²⁺ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca²⁺ channels on the t-tubule and SR membranes.

Ca²⁺ itself or Ca²⁺-dependent signaling pathways play fundamental roles in various cellular processes from cell growth to death. The most representative example can be found in skeletal muscle cells where a well-timed and adequate supply of Ca²⁺ is required for coordinated Ca²⁺-dependent skeletal muscle functions, such as the interactions of contractile proteins during contraction. Intracellular Ca²⁺ movements between the cytosol and sarcoplasmic reticulum (SR) are strictly regulated to maintain the appropriate Ca²⁺ supply in skeletal muscle cells. Added to intracellular Ca²⁺ movements, the contribution of extracellular Ca²⁺ entry to skeletal muscle functions and its significance have been continuously studied since the early 1990s. Here, studies on the roles of channel proteins that mediate extracellular Ca²⁺ entry into skeletal muscle cells using skeletal myoblasts, myotubes, fibers, tissue, or skeletal muscle-originated cell lines are reviewed with special attention to the proposed functions of transient receptor potential canonical proteins (TRPCs) as store-operated Ca²⁺ entry (SOCE) channels under normal conditions and the potential abnormal properties of TRPCs in muscle diseases such as Duchenne muscular dystrophy (DMD).

Calsequestrin (CASQ) was discovered in rabbit skeletal muscle tissues in 1971 and has been considered simply a passive Ca²⁺-buffering protein in the sarcoplasmic reticulum (SR) that provides Ca²⁺ions for various Ca²⁺signals. For the past three decades, physiologists, biochemists, and structural biologists have examined the roles of the skeletal muscle type of CASQ (CASQ1) in skeletal muscle and revealed that CASQ1 has various important functions as (1) a major Ca²⁺-buffering protein to maintain the SR with a suitable amount of Ca²⁺at each moment, (2) a dynamic Ca²⁺sensor in the SR that regulates Ca²⁺release from the SR to the cytosol, (3) a structural regulator for the proper formation of terminal cisternae, (4) a reverse-directional regulator of extracellular Ca²⁺entries, and (5) a cause of human skeletal muscle diseases. This review is focused on understanding these functions of CASQ1 in the physiological or pathophysiological status of skeletal muscle.