Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition.
The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem. 269, 26598-26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry 36, 11865-11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+ transport. A single mutation in the S3 stalk segment (Gly257 --> Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor.[1]References
- Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition. Zhong, L., Inesi, G. J. Biol. Chem. (1998) [Pubmed]
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