Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b

Alan R. Penheiter, Ariel J. Caride, A. Enyedi, John T. Penniston

Research output: Contribution to journalArticle

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Abstract

Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca2+ pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was ∼2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K0.5 of 9 nM for wild type to 144 nM (assayed at 700 nM free Ca2+). Pre-steady state measurements of the rate of activation by Ca2+-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the half-time of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca2+-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.

Original languageEnglish
Pages (from-to)17728-17732
Number of pages5
JournalJournal of Biological Chemistry
Volume277
Issue number20
DOIs
Publication statusPublished - May 17 2002

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Calmodulin
Cell membranes
Tryptophan
Cell Membrane
Pumps
Mutation
Chemical activation
Peptides
COS Cells
Alanine
Protein Isoforms
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b. / Penheiter, Alan R.; Caride, Ariel J.; Enyedi, A.; Penniston, John T.

In: Journal of Biological Chemistry, Vol. 277, No. 20, 17.05.2002, p. 17728-17732.

Research output: Contribution to journalArticle

Penheiter, Alan R. ; Caride, Ariel J. ; Enyedi, A. ; Penniston, John T. / Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 20. pp. 17728-17732.
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