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Mitsugumin 23 (MG23) has been identified as a ball-shaped cation channel in the sarcoplasmic reticulum (SR) but its physiological role remains unclear. This study aimed to examine the contribution of MG23 to Ca2+ storage function in skeletal muscle by using Mg23-knockout (Mg23-/-) mice. There was no difference in the isometric specific force of the extensor digitorum longus (EDL) and soleus (SOL) muscles between Mg23-/- and wild-type (Wt) mice. In Mg23-/- mice, the calsequestrin 2 content in the EDL muscle and SR Ca2+-ATPase 2 content in the SOL were increased. We have examined SR and myofibril functions using mechanically skinned fibers and determined their fiber types based on the response to Sr2+, which showed that Mg23-/- mice, compared with Wt, had: 1) elevated total Ca2+ content in the membranous components including SR, mitochondria, and transverse tubular system referred to as endogenous Ca2+ content, in both type I and II fibers of the EDL and SOL; 2) increased maximal Ca2+ content in both type I and II fibers of the EDL and SOL; 3) decreased SR Ca2+ leakage in type I fibers of the SOL; and 4) enhanced SR Ca2+ uptake in type I fibers of the SOL, although myofibril function was not different in both type I and II fibers of the SOL and EDL muscles. These results suggest that MG23 decreases SR Ca2+ storage in both type I and type II fibers, likely due to increased SR Ca2+ leakage. [ABSTRACT FROM AUTHOR]