Study of calcium influx dynamics in erythrocytes treated with E.coli alpha hemolysin

ROMINA VAZQUEZ; SABINA MATÉ; CARLOS MUÑOZ GARAY; LAURA BAKÁS; VANESA HERLAX

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Alpha hemolysin (HlyA) is an exotoxin secreted by uropathogenic E. coli strains that targets many cell types causing lysis and death. HlyA is claimed to be a pore forming toxin although no pore structure has been identified yet. After treating erythrocytes with HlyA a Ca2+ influx occurs that ultimately leads to cell swelling and osmotic lysis. Recently it has been shown that Ca2+ influx initially triggers erythrocytes shrinkage through activation of K3.1 channels and subsequent K+ effux1. It is not well established if initial Ca2+ entry occurs through the toxin pore or through cation channels. Nevertheless, it has been demonstrated that purinergic channels amplificate the signal2. Ca2+ entry in rabbit erythrocytes treated with HlyA shows a biphasic behavior, with a first slight increase followed by a second increment that keeps rising until lysis occurs3. The aim of the present work was to further explore the mechanism of Ca2+ entry. Inhibition of Ca2+ channels with Ni+2 or Brilliant Blue G impaired HlyA hemolytic activity in rabbit erythrocytes. Nevertheless, when Ca2+ influx was monitored by Fluo-4 fluorescence imaging of individual cells, the initial increase in Ca2+ concentration was still observed. In order to test if this Ca2+ increment was a result of toxin insertion into the membrane, Ca+2 levels were monitored in erythrocytes treated with proHlyA, the unacylated nonhemolytic form of HlyA. ProHlyA showed insertion levels similar to those of HlyA in lipid monolayers mimicking the outer leaflet of erythrocytes membranes but did not cause Ca2+ influx in erythrocytes. Strikingly, cell shrinkage was still observed. These results suggest that monomer toxin insertion into the membrane may trigger cellular responses leading to cell shrinkage independently of Ca2+ levels. Nevertheless, acyl chains covalently bound to the toxin are necessary for initial Ca2+ entry and amplification through Ca2+ channels activation is further needed for lysis.