Effects of auxiliary subunits and lipid environment on plasma membrane calcium ATPases

BRUNO, CAMILA; SOUTO GUEVARA, CECILIA; FERREIRA GOMES, MARIELA; ROSSI JUAN PABLO; MANGIALAVORI IRENE

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

The maintenance of calcium homeostasis is essential for the normal functioning of cells,as it´s involved in various processes such as muscle contraction, exocytosis, andapoptosis. Plasma membrane calcium ATPase (PMCA) is one of the proteins involved in theregulation of calcium homeostasis. It belongs to the P-ATPase family and uses ATPhydrolysis as energy source to mediate calcium´s transport from the intracellular to theextracellular environment against an electrochemical gradient. PMCA has a C-terminaldomain 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 autoinhibitoryinteraction, placing the pump in an open conformation. PMCA is a complex protein thathas an additional subunit that can be either Basigin or Neuroplastin, depending on theexpression in each tissue. This subunit contributes to the stability and correct localizationof the protein in the plasma membrane. It has been suggested that either Basigin orNeuroplastin is required for the Ca2+-ATPase activity of the pump.With the aim of determining whether the additional subunit of PMCA is required forcalcium transport, we assessed the effects of Basigin and different lipid environments onPMCA activity and structure: (1) By means of a functional approach, we determined theapparent affinity of the pump for calcium and calmodulin´s activation in humanerythrocyte membranes, in the presence of Basigin, and in the purified enzymereconstituted in different lipid surroundings; (2) Using structural methods, we identifiedproteinase K-controlled proteolysis and distinct intrinsic fluorescence of PMCA afterrecombination in a lipid environment to assess the accessibility of the C-terminalautoinhibitory domain.Our results show that PMCA is active in the absence of its subunits and that the apparentaffinity of calcium does not change in the presence or absence of Basigin. On the otherhand, PMCA activity is strongly influenced by the biophysical properties of the lipidenvironment. The accessibility of the C-terminal domain and the intrinsic fluorescence ofthe pump are sensitive to the lipid environment. With all these results, this work is the firststep in trying to clarify the relationship between PMCA structure and function for propercalcium transport, and cooperative maintenance of calcium homeostasis.AcknowldegmentsThis work was supported by ANCyT, Universidad de Buenos Aires and CONICET82