CONICET | Buscador de Institutos y Recursos Humanos

The maintenance of calcium homeostasis is essential for the normal functioning of cells, as it is involved in various processes such as muscle contraction, exocytosis, and apoptosis. Plasma membrane calcium ATPase (PMCA) is one of the proteins involved in the regulation of calcium homeostasis. It belongs to the P-ATPase family and uses ATP hydrolysis as energy source to mediate the transport of calcium from the intracellular to the extracellular environment against an electrochemical gradient. PMCA has a C-terminal domain that interacts with the catalytic region of the pump, providing a partially active ?closed? conformation. Maximal activity is achieved when calcium levels rise and the Ca2-CaM complex can bind to the C-terminus of PMCA, disrupting the autoinhibitory interaction and placing the pump in an open conformation. PMCA is a complex protein that has an additional subunit that can be either Basigin or Neuroplastin, depending on the expression in each tissue . This subunit contributes to the stability and correct localization of the protein in the plasma membrane. It has even been suggested that either Basigin or Neuroplastin is required for the Ca2-ATPase activity of the pump . With the aim of determining whether the additional subunit of PMCA is required for calcium transport, we assessed the effects of Basigin and different lipid environments on PMCA activity and structure: (1) By means of a functional approach, we determined the apparent affinity of the pump for calcium and calmodulin´s activation in human erythrocyte membranes, in the presence of Basigin, and in the purified enzyme reconstituted in different lipid surroundings; (2) Using structural methods, we identified proteinase K-controlled proteolysis and distinct intrinsic fluorescence of PMCA after recombination in a lipid environment to assess the accessibility of the C-terminal autoinhibitory domain.Our results show that PMCA is active in the absence of its subunits and that the apparent affinity of calcium does not change in the presence or absence of Basigin. On the other hand, PMCA activity is strongly influenced by the biophysical properties of the lipid environment. The accessibility of the C-terminal domain and the intrinsic fluorescence of the pump are sensitive to the lipid environment. With all these results, this work is the first step in trying to clarify the relationship between PMCA structure and function for proper calcium transport, and cooperative maintenance of calcium homeostasis. This work was supported by ANCyT, Universidad de Buenos Aires and CONICET