Involvement of the “A” Domain in the autoinhibition of the Plasma Membrane Ca2+ Pump

MAZZITELLI L.R.; ADAMO HP

Buzios, Brasil

Workshop; First Latin American PostGraduate Program Course; 2009

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0pt; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 85.05pt 70.85pt 85.05pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> It has been proposed that the autoinhibition of the Ca2+ pump of plasma membrane (PMCA) involves the blockage of the active site of the enzyme by the C-terminal segment. Ca2+-calmodulin binds to the C-terminal segment and stops inhibition.  Recent studies in other members of the P-type ATPase family indicate that the A domain controls the access to the transport site which is buried in the transmembrane region of the molecule. The amino acid segment Ile206-Val271 on the top of A domain of the PMCA has been directly implicated in the interaction with the C-terminal segment. However, studies of other autoinhibited pumps, suggest that autoinhibition can be controlled by a structure at the bottom of domain A formed by the residues that connect this domain with the transmembrane segments. We have investigated the functional effect of domain A substitutions Ile227Ala, Asp223Ala, and Glu99Gln, this latter residue located in the connecting stretch between domain A and transmembrane segment M1. Substitutions Ile227Ala and Asp223Ala led decreased the maximal activity to 45 % and 10 %, respectively. In contrast the maximal activity of mutant Glu99Gln was equal or higher than that of the wild type enzyme. In the absence of calmodulin the activity of the Ile227Ala mutant was reduced to 66% of the maximal activity suggesting that autoinhibition was taking place. While mutants at Ile227 and Asp223 reduced the activity of the enzyme probably by altering the structure of domain “A”,  Glu99   together with residue Asp170 identified previously (Bredeston and Adamo, 2004)  may be part of a negatively charged structure required for  autoinhibition of the PMCA. (With Grants from UBA, CONICET and ANPCyT Prestamo BID PICT 15-25965)