Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum.

摘要: Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs expressed in COS-1 cells, and Ca2+-ATPases assayed for Ca2+ transport function phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, 3 resulted partial loss function. In most cases, subsequent mutation individual amino acids cluster had no effect on activity. one cluster, however, it was possible to assign reduction activity alterations Asn111 Asn114. Asn114 alanine retained about 50% activity, whereas change only 10% None affected phosphorylation enzyme ATP presence inorganic phosphate absence Ca2+. combined experiments suggest that reduced uptake observed mutants not due a defect activation formation phosphorylated intermediate but rather incompetent handling bound following utilization. These results demonstrate acidic amidated region do constitute high affinity Ca2+-binding sites whose occupancy is required activation. They may, act sequester cytoplasmic channel domains are involved cation translocation. Simultaneous 4 glutamate lumenal loop between transmembrane sequences M1 M2 did affect indicating this play an essential role transport. Similarly, Glu192 Asp196 beta-strand domain helices 2 although diminish expression protein.