A model for the activation of plasma membrane calcium pump isoform 4b by calmodulin.
Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.
Penheiter
Alan R
AR
Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901, USA.
Bajzer
Zeljko
Z
Filoteo
Adelaida G
AG
Thorogate
Richard
R
Török
Katalin
K
Caride
Ariel J
AJ
D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D027682:Cation Transport Proteins; D004789:Enzyme Activation; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014227:Triazines / Q000737:chemistry / Q000378:metabolism
C404210:2-chloro-(6-(4-N,N-diethylaminophenyl)-1,4,5-triazin-4-yl)calmodulin; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D027682:Cation Transport Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D014227:Triazines; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases