Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

• Published in: The Journal of biological chemistry 2002-05, Vol.277 (20), p.17728-17732
• Main Authors: Penheiter, Alan R., Caride, Ariel J., Enyedi, Ágnes, Penniston, John T.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calcium - metabolism; Calcium Channels - genetics; Calcium Channels - metabolism; Calcium-Transporting ATPases - metabolism; Calmodulin - metabolism; Cation Transport Proteins; COS Cells; Enzyme Activation; Kinetics; Molecular Sequence Data; Plasma Membrane Calcium-Transporting ATPases; Structure-Activity Relationship; Tryptophan - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111608200

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.