Mitsugumin 29 (MG29) is related to the fatigue and aging processes of skeletal muscle. To examine the roles of MG29 in conjunction with its binding protein, the canonical-type transient receptor potential cation channel 3 (TRPC3), in skeletal muscle, the binding region of MG29 to TRPC3 was studied along with the functional relevance of the binding in mouse primary skeletal myotubes using coimmunoprecipitation assays and Ca2+ imaging experiments. The N-terminus and the I-II loop of MG29 constitute the binding region for TRPC3. The myotubes that expressed the MG29 mutant missing the entire TRPC3-binding region showed a disrupted binding between endogenous MG29 and TRPC3 and a reduction in Ca2+ transients in response to membrane depolarization without affecting ryanodine receptor 1 activity, the resting cytosolic Ca2+ level, and the amount of releasable Ca2+ from the sarcoplasmic reticulum. Among the proteins mediating Ca2+ movements in skeletal muscle, TRPC4 expression was significantly decreased by the MG29 mutant. Therefore, MG29 could be a new factor for regulating Ca2+ transients during skeletal muscle contraction possibly via a correlation with TRPC3 and TRPC4. (C) 2015 Elsevier Inc. All rights reserved.