Effect of Entomocidal Proteins from Bacillus thuringiensis on Ion Permeability of Apical Membranes of Tenebrio molitor Larvae Gut Epithelium

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I. M. Andreev^1*, N. V. Bulushova², I. A. Zalunin², and G. G. Chestukhina²

¹Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, 127276 Moscow, Russia; fax: (495) 977-8018; E-mail: pmembrane@ippras.ru

²Institute of Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 117545 Moscow, Russia; fax: (495) 315-0501; E-mail: zalunin@genetika.ru

^* To whom correspondence should be addressed.

Received March 19, 2009; Revision received April 20, 2009

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Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and ΔpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg²⁺ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg²⁺ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO₄ or CaCl₂ in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca²⁺-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K₂SO₄. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K₂SO₄ exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.

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KEY WORDS: Bacillus thuringiensis, δ-endotoxin Cry3A, Tenebrio molitor, BBMV, membrane potential, ion channels, potential-sensitive dyes, membranotropic effect of δ-endotoxin Cry3A

DOI: 10.1134/S0006297909100058

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