SPECIFIC SUBSTITUTIONS AT AMINO-ACID-256 OF THE SARCOPLASMIC ENDOPLASMIC RETICULUM CA2+ TRANSPORT ATPASE MEDIATE RESISTANCE TO THAPSIGARGININ THAPSIGARGIN-RESISTANT HAMSTER-CELLS/

High levels of resistance to thapsigargin (TG), a specific inhibitor o f intracellular Ca2+ transport ATPases (SERCAs), can be developed in c ulture by stepwise exposure of mammalian cells to increasing concentra tions of TG. We have identified, in two independently selected TG-resi stant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/T G, a T --> C change at nucleotide 766 introduces a Phe(256) --> Leu al teration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 mu M , a T --> C change at nucleotide 767 introduces a Phe(256) --> Ser mut ation at that position. When these specific mutations are introduced i nto a wild-type full-length avian SERCA1 cDNA, transfection experiment s reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations, However, a 4-5-fold resistance to TG in hibition of Ca2+ transport function occurs upon the introduction of ei ther the Phe(256) --> Leu or the Phe(256) --> Ser mutation into wild-t ype SERCA1, These specific mutations also render the hydrolytic activi ty of the ATPase resistant to inhibition by TG, Our results not only i mplicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.