Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b.
Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) 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 approximately 2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K(0.5) of 9 nm for wild type to 144 nm (assayed at 700 nm free Ca(2+)). Pre-steady state measurements of the rate of activation by Ca(2+)-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 Ca(2+)-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.
Penheiter
Alan R
AR
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Caride
Ariel J
AJ
Enyedi
Agnes
A
Penniston
John T
JT
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D027682:Cation Transport Proteins; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D053499:Plasma Membrane Calcium-Transporting ATPases; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000502:physiology*
D015220:Calcium Channels; D002147:Calmodulin; D027682:Cation Transport Proteins; D014364:Tryptophan; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium