No abstract

Four male patients from two families were first seen with impaired skeletal muscle relaxation that rapidly worsened during exercise. Muscle biopsies from 2 patients were examined by appropriate biochemical and microscopic immunocytochemical techniques. The adenosine triphosphate (ATP)-dependent Ca2+ transport rate was extremely low in a particulate membrane fraction of skeletal muscle, and there was also a marked reduction of the concentration of 100-kD phosphoprotein, corresponding to Ca2+-ATPase of sarcoplasmic reticulum, in muscle microsomes. The concentration of immunoreactive Ca2+-ATPase of sarcoplasmic reticulum was markedly reduced on immunoblots. Evaluation by microscopic immunocytochemical techniques, using one polyclonal and two monoclonal antibodies against sarcoplasmic reticulum Ca2+ transport protein, revealed that the severe reduction of immunoreactive Ca2+-ATPase was limited to the histochemical type 2 fibers. The deficiency of the Ca2+ transport protein in the sarcoplasmic reticulum of type 2 fibers, which may be the primary expression of a presumed gene defect, can explain the impaired muscle relaxation of the patients. This disease appears to be a clinically, electromyographically, and biochemically distinct metabolic myopathy.

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2 . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."Muscle contraction is triggered by the release ofCa2l from the sarcoplasmic reticulum, whereas muscle relaxation is achieved by rapid reuptake of Ca2l from the cytosol into the lumen ofthe sarcoplasmic reticulum. Sequestration of Ca2l in the lumen of the sarcoplasmic reticulum is also an essential step in the overall contraction-relaxation cycle of muscle cells. The Ca2' pump has been found to be distributed along the membranes ofthe longitudinal sarcoplasmic reticulum and the nonjunctional region of the terminal cisternae (1), whereas the Ca+ release channel from sarcoplasmic reticulum has been localized in the junctional face of the terminal cisternae (1, 2). The low-affinity, high-capacity Ca2+ sequestering protein, calsequestrin, is located in the lumen of the terminal cisternae (3-5).The sarcoplasmic reticulum also contains two immunochemically related glycoproteins of unknown function, with apparent molecular masses of 53 and 160 kDa (6, 7). It has been proposed (8, 9) that the glycoproteins are involved in the regulation of Ca2+ transport. We have cloned cDNA encoding the 53-kDa glycoprotein (7) but were unable to deduce its function from analysis of its primary structure. In this study we describe the cloning of cDNA encoding the 160-kDa glycoprotein. ¶ Analysis of its deduced amino acid sequence and studies of its localization and Ca2" binding properties lead us to conclude that it is a Ca2+ binding protein located in the lumen of the longitudinal sarcoplasmic reticulum.MATERIALS AND METHODS Isolation, Analysis, and Expression of cDNA Clones. A Agtll cDNA expression library from rabbit fast-twitch skeletal muscle (a kind gift from

We report a family in which four members in two generations (mother, her son, and two daughters) suffered from impaired muscle relaxation aggravated by exercise. Muscle biopsies from two sisters showed moderate degree of histochemical type 2 fiber atrophy and excess of internal nuclei. Microscopic immunocytochemistry, using a monoclonal antibody raised against purified chicken SR-ATPase, revealed severe reduction of the immunoreactive ATPase of SR was markedly decreased on Western blots of muscle proteins. This family appears to have a clinically, electromyographically, and biochemically distinct metabolic myopathy associated with deficiency of SR-ATPase, with a probable autosomal dominant inheritance pattern that is phenotypically similar to recently described recessive cases.