Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites.
The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.
Andersen
J P
JP
Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark. jpa@fi.au.dk
Sorensen
T L
TL
Povlsen
K
K
Vilsen
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium