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 Ca(2+)-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 Ca(2+). Furthermore, Na(+) and Ca(2+) 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(+)/Ca(2+)-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca(2+)-binding sequence in the protease acted as a Na(+) sensor. Proteomic analyses using Cs(+)/Mg(2+) and p94CS-KI mice as negative controls revealed that Na(+) and Ca(2+) 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 [Ca(2+)], 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.
Ono
Yasuko
Y
Calpain Project, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 156-8506, Japan.
Ojima
Koichi
K
Torii
Fukuyo
F
Takaya
Emi
E
Doi
Naoko
N
Nakagawa
Kazuhiro
K
Hata
Shoji
S
Abe
Keiko
K
Sorimachi
Hiroyuki
H
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D009154:Mutation; D009928:Organ Specificity; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity
D012964:Sodium; D002154:Calpain; C078134:calpain p94; D002118:Calcium
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