Staphylococcus aureus alpha-toxin permeabilizes the basolateral membrane of a Cl(-)-secreting epithelium.
Apical membrane ion channels control the rate of transepithelial electrolyte transport in many epithelia. One way to study such channels in their native location, the apical membrane, is to eliminate the resistance of the basolateral membrane to ion flow. Then the opening and closing of apical channels can be measured as a transepithelial current, free from the influence of basolateral membrane transport processes. To develop a method that would permeabilize an epithelial basolateral membrane to ions and nucleotides, we examined the effect of Staphylococcus aureus alpha-toxin on the Cl(-)-secreting T84 epithelial cell line. alpha-Toxin permeabilized the basolateral, but not the apical membrane to Cl-, adenosine 3',5'-cyclic monophosphate (cAMP), and GTP. However, the integrity of signal-transduction pathways, the regulation of apical membrane Cl- channels, and the transepithelial resistance remained intact. In the course of examining the effect of ATP, we found that the basolateral membrane contained purinergic receptors that both stimulated Cl- secretion on their own and, at high concentrations, inhibited cAMP-induced Cl- secretion. These effects of extracellular ATP were eliminated after prolonged exposure to ATP, suggesting receptor downregulation. In addition, depletion of intracellular ATP following permeabilization prevented cAMP-dependent regulation of apical Cl- channels. We conclude that alpha-toxin may prove to be a useful tool for studying the regulation and properties of apical membrane ion channels.
Ostedgaard
L S
LS
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Shasby
D M
DM
Welsh
M J
MJ
D000255:Adenosine Triphosphate / Q000172:deficiency; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D013208:Staphylococcal Toxoid / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002712:Chlorides; D013208:Staphylococcal Toxoid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP