title: Hyperactivation of the human plasma membrane Ca2+ pump PMCA h4xb by mutation of Glu99 to Lys.
abstract: The transport of calcium to the extracellular space carried out by plasma membrane Ca(2+) pumps (PMCAs) is essential for maintaining low Ca(2+) concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca(2+)-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca(2+). Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the Vmax. Mutation of residue Glu(99) to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the Vmax of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca(2+). Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Luciana R
authors.initials: LR
authors.lastname: Mazzitelli
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina. Electronic address: hpadamo@qb.ffyb.uba.ar.
authors.forename: Hugo P
authors.initials: HP
authors.lastname: Adamo
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017353:Gene Deletion; D005973:Glutamine / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: C506952:ATP2B1 protein, human; D002147:Calmodulin; D020033:Protein Isoforms; D005973:Glutamine; D049452:Green Fluorescent Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium
keywords: ATPases; Calcium ATPase; Calcium Transport; Enzymes; Transporters