Transmembrane segment M3 is essential to thapsigargin sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase.
Five different chimeric fusion proteins between the sarcoplasmic reticulum Ca(2+)-ATPase and the rat kidney alpha 1 isoform of the Na+,K(+)-ATPase were expressed in COS-1 cells and analyzed functionally. In two chimeras, the only Na+,K(+)-ATPase-derived part consisted of the region corresponding to the third transmembrane segment. These proteins phosphorylated from ATP in the presence of a high Ca2+ concentration and from Pi in the absence of Ca2+, but the phosphorylation displayed strongly reduced sensitivity to inhibition by thapsigargin. In two other chimeras, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region containing the fourth transmembrane segment. These proteins were unable to phosphorylate from ATP but phosphorylated from Pi, the latter reaction being insensitive to inhibition by thapsigargin. In the last chimera, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region corresponding to both the third and the fourth transmembrane segments. This chimera phosphorylated from Pi in the reaction that was fully sensitive to inhibition by thapsigargin. It can be concluded that the third transmembrane segment is indispensable to thapsigargin sensitivity of the Ca(2+)-ATPase and most likely, therefore, constitutes a major binding region for thapsigargin.
Nørregaard
A
A
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
Vilsen
B
B
Andersen
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D007668:Kidney / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology*; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014162:Transfection
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013729:Terpenes; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium