title: Formation of very large conductance channels by Bacillus cereus Nhe in Vero and GH(4) cells identifies NheA + B as the inherent pore-forming structure.
abstract: The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH(4)) by measuring release of lactate dehydrogenase (LDH), K(+) efflux and the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K(+) as well as Ca(2+) influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca(2+)](i). Exposure to NheA + B caused a similar K(+) efflux and elevation of [Ca(2+)](i) as NheA + B + C in GH(4) cells, whereas in Vero cells the rate of K(+) efflux was reduced by 50% and [Ca(2+)](i) was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH(4) cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH(4) cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH(4) cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, Post Box 1041, 0316 Oslo, Norway. t.m.haug@imbv.uio.no
authors.forename: Trude M
authors.initials: TM
authors.lastname: Haug
authors.forename: Sverre L
authors.initials: SL
authors.lastname: Sand
authors.forename: Olav
authors.initials: O
authors.lastname: Sand
authors.forename: Danh
authors.initials: D
authors.lastname: Phung
authors.forename: Per E
authors.initials: PE
authors.lastname: Granum
authors.forename: Simon P
authors.initials: SP
authors.lastname: Hardy
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000378:metabolism*; D002118:Calcium; D002460:Cell Line; D002522:Chlorocebus aethiops; D004594:Electrophysiology; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D051381:Rats; D014709:Vero Cells
chemical_list: D004768:Enterotoxins; D051036:Large-Conductance Calcium-Activated Potassium Channels; C040792:enterotoxin, Bacillus cereus; D011188:Potassium; D002118:Calcium
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