Vibrio vulnificus Cytolysin Forms Anion-selective Pores on the CPAE Cells, a Pulmonary Endothelial Cell Line

Abstract

Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of 37.5±4.0 pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of Na+ in bath solution with monovalent ions (K+, Cs+ or TEA+) or with divalent ions (Mg2+, Ca2+) did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to -11.8 and -28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at -40 mV was Cl- = NO2- ≥ Br- = I- ＞ SCN- ＞ acetate- ＞ isethionate- ＞ ascorbic acid- ＞ EDTA2-, in descending order. The cytolysin-induced pore current was blocked by Cl- channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells