Erythrocyte plasma membrane potential

BALACH, MELISA MICAELA; MONESTEROLO, NOELIA E.; SANTANDER, VERÓNICA S.; CASALE, CESAR H.; CAMPETELLI, ALEXIS N.

Congreso; YIB Talks (Jóvenes biofísicos, The Young Iniciative on Biophysics ); 2020

jóvenes Biofísicos "the Young Iniciative on Biophysics"

The plasma membrane functions both as a natural insulator and a semi-permeable barrier to the movement of ions. A wide variety of proteins transport and pump ions to generate concentration gradients that result in voltage differences, while ion channels allow ions to move across the membrane down those gradients. Plasma membrane potential (PMv) is the difference in voltage between the inside and the outside of a cell, and it ranges from ~− 3 to ~− 90 mV. The most significant discoveries in this field have been made in excitable cells, nevertheless, special attention has been paid to some events controlled by changes in membrane potential in non-excitable cells. The origins of several blood disorders, for instance, are related to disturbances at the level of erythrocyte plasma membrane. Given their simplicity, erythrocytes have been perfect candidates for the electrophysiological studies that laid the foundations for understanding the generation, maintenance, and roles of membrane potential. Various methodologies have been used during the last decades to determine membrane potential in red blood cells, the use of microelectrodes, nuclear magnetic resonance, the use of lipophilic radioactive ions to quantify intra and extracellular ions, as well as fluorescent potentiometric dyes that are continuously renewed and upgraded. Our group showed that erythrocytes from diabetic and hypertensive patients displayed more tubulin associated with the plasma membrane than erythrocytes from normal subjects. This association results in the inhibition of plasma membrane P-ATPases, as Na+,K+-ATPase and Ca2+-ATPase. Consequently, the intracellular concentration of Na+ and Ca+2 increases. By taking into account that tubulin has a negative charge at cytoplasmic pH, and using biochemical tools and the eletric-sensitive fluorescent dye DiBAC4(3) we designed experiments to know whether changes in the PMv could influence the migration of tubulin to the plasma membrane. For this, we fine-tuned the determination of PMv by using severalfluorescence quantification systems as fluorescence microscopy, spectrofluorometry and flow cytometry. Among these techniques, flow cytometry resulted more advantageous to perform the experiments since it significantly reduced the number of drugs and cells to be used, as well as the measurement times, and in addition, we obtained fluorescence values with lower standard deviations. Finally, we determined the PMv in erythrocytes from diabetic and hypertensive patients, which were depolarized, when compared with healthy cells, thus correlating the amount of tubulin associated with the membrane with cellular PMv.