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

• Published in: The Journal of membrane biology Vol. 237; no. 1; pp. 1 - 11
• Main Authors: Haug, Trude M, Sand, Sverre L, Sand, Olav, Phung, Danh, Granum, Per E, Hardy, Simon P
• Format: Journal Article
• Language: English
• Published: United States 01-09-2010
• Subjects: Animals; Bacillus cereus - metabolism; Calcium; Cell Line; Chlorocebus aethiops; Electrophysiology; Enterotoxins - genetics; Enterotoxins - metabolism; Large-Conductance Calcium-Activated Potassium Channels - genetics; Large-Conductance Calcium-Activated Potassium Channels - metabolism; Potassium - metabolism; Rats; Vero Cells
• ISSN: 1432-1424
• DOI: 10.1007/s00232-010-9298-6

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.