Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Because intracellular [Na⁺] is kept low by Na⁺/K⁺-ATPase, Na⁺ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca²⁺-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na⁺-dependent, but not Cs⁺-dependent, autolysis in the absence of Ca²⁺. Furthermore, Na⁺ and Ca²⁺ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na⁺/K⁺-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na⁺/Ca²⁺-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca²⁺-binding sequence in the protease acted as a Na⁺ sensor. Proteomic analyses using Cs⁺/Mg²⁺ and p94CS-KI mice as negative controls revealed that Na⁺ and Ca²⁺ direct p94 to proteolyze different substrates. We propose three roles for Na⁺ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca²⁺], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na⁺-dependent enzyme.