Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.
Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising the Ca2+ concentration to the mM range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+.
Strock
C
C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Cavagna
M
M
Peiffer
W E
WE
Sumbilla
C
C
Lewis
D
D
Inesi
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000201:enzymology; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection / Q000235:genetics
D002135:Calcium-Binding Proteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium