Erythrocytes potential membrane

MONESTEROLO NE; CAMPETELLI AN; BALACH MM; CASALE CH; SANTANDER VS

Congreso; #YIB #TheYoungIniciativeonBiophysics #JovenesBiofisicos; 2020

Sociedad Argentina de Biofisica

The plasma membrane functions both as a natural insulator and a semi-permeable barrier to themovement of ions. A wide variety of proteins transport and pump ions to generate concentrationgradients that result in voltage differences, while ion channels allow ions to move across themembrane down those gradients. Plasma membrane potential (PMv) is the difference in voltagebetween the inside and the outside of a cell, and it ranges from ~− 3 to ~− 90 mV. Most of themost significant discoveries in this field have been made in excitable cells, nevertheless, specialattention has been paid to some events controlled by changes in membrane potential in nonexcitablecells. The origins of several blood disorders, for instance, are related to disturbances atthe level of plasma membrane in erythrocytes. Given their simplicity, erythrocytes have beenperfect candidates for the electrophysiological studies that laid the foundations for understandingthe generation, maintenance, and roles of membrane potential. Various methodologies have beenused during the last decades to determine membrane potential in red blood cells, the use ofmicroelectrodes, nuclear magnetic resonance, the use of lipophilic radioactive ions to quantifyintra and extracellular ions, as well as fluorescent potentiometric dyes that are continuouslyrenewed and upgraded. Our group showed that erythrocytes from diabetic and hypertensivepatients displayed more tubulin associated with the plasma membrane than erythrocytes fromnormal subjects. This association results in the inhibition of plasma membrane P-ATPases, asNa+,K+-ATPase and Ca2+-ATPase. Consequently, the intracellular concentrations of Na+ andCa+2 increases. By taking into account that tubulin has a negative charge at cytoplasmic pH, andusing biochemical tools and the eletric-sensitive fluorescent dye DiBAC4(3) we designedexperiments to know whether changes in the PMv could influence the migration of tubulin to theplasma membrane. For this, we fine-tuned the determination of PMv by using several fluorescencequantification systems as fluorescence microscopy, spectrofluorometry and flow cytometry.Among these techniques, flow cytometry resulted more advantageous to perform theexperiments since it significantly reduced the number of drugs and cells to be used, as well as themeasurement times, and in addition, we obtained fluorescence values with lower standarddeviations. Finally, we determined the PMv in erythrocytes from diabetic and hypertensivepatients, wich were, depolarized, when compared with healthy cells, thus correlating the amountof tubulin associated with the membrane with cellular PMv.