High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca 2؉ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T 3 C change at nucleotide 766 introduces a Phe 256 3 Leu alteration

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... n the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4M, a T 3 C change at nucleotide 767 introduces a Phe 256 3 Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca 2؉ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4 -5-fold resistance to TG inhibition of Ca 2؉ transport function occurs upon the introduction of either the Phe 256 3 Leu or the Phe 256 3 Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.