Location of high affinity Ca^{2 +}-binding sites within the predicted transmembrahe domain of the sarco-plasmic reticulum Ca²⁺-ATPase

Abstract

CATION pumps bind and translocate ions with the intermediate formation of a phosphoenzyme. In spite of extensive knowledge of the primary and even secondary structures of several of these cation transport enzymes, however, no high affinity cation binding sites have yet been determined. Here we report the use of oligo-nucleotide-directed, site-specific mutagenesis to identify the amino acids involved in Ca²⁺ binding in one of these transport enzymes, the Ca²⁺-ATPase of sarcoplasmic reticulum. Alteration of Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 or Glu 908, each of which is predicted to lie near the centre of the transmembrane domain in putative transmembrane sequences M₄, M₅, M₆ and M₈^1–3 resulted in complete loss of Ca²⁺ transport function and of Ca²⁺-dependent phosphorylation of the enzyme by ATP. Phosphorylation of each of the mutant enzymes with inorganic phosphate was observed, however, even in the presence of Ca²⁺, which inhibits phosphorylation in the wild-type enzyme possessing an intact high affinity Ca²⁺-binding site. These results suggest that at least six polar, oxygen-containing residues lying near the centre of the transmembrane domain provide ligands for one or both of the two high affinity Ca²⁺ binding sites in the Ca²⁺-ATPase.