Patch-clamp study of the apical membrane of the midgut of Manduca sexta larvae: direct demonstration of endogenous channels and effect of a Bacillus thuringiensis toxin
Introduction
The midgut of lepidopteran larvae is a K+-secreting epithelium composed of two major cell types, the goblet and columnar cells, which appear to be electrically coupled (Klein et al., 1996). The pear-shaped goblet cells are responsible for strong active K+ secretion from haemolymph to lumen (Harvey et al., 1983, Dow and Harvey, 1988) and for large K+ and pH gradients across the midgut, while the columnar cells are absorptive cells with a well-developed apical brush-border membrane. Briefly, K+ secretion is mediated by a vacuolar-type proton ATPase (V-ATPase) coupled with an electrogenic K+/2H+ exchanger, both located in the apical membrane of goblet cells (Wieczorek et al., 1989, Wieczorek et al., 1991, Wieczorek et al., 1999). This process maintains a large potential difference in the order of 150 mV across the epithelium. The K+ electrochemical gradient generated across the apical membrane is used for nutrient uptake into the columnar cells via apical K+/amino acid symporters (Giordana et al., 1989). While the route of basolateral K+ entry in midgut epithelial cells is mainly via Ba2+-sensitive channels (Zeiske et al., 1986, Moffett and Lewis, 1990), the apical exit pathway of K+ is strictly confined to the goblet cells via their apical valves. Recently, a few studies have demonstrated changes in midgut active ion transport, V-ATPase activity and metabolism during larval moulting or starvation (Sumner et al., 1995, Gräf et al., 1996, Chamberlin et al., 1997, Chamberlin and King, 1998).
In addition to its physiological role in K+ transport and absorption of nutrients, the midgut is the target organ of, or at least the first barrier to be crossed by, numerous microbial pathogens, natural toxins and chemical insecticides that act orally. Specifically, the apical brush-border membrane of the epithelial cells that line the insect midgut lumen is the main target of the pore-forming Bacillus thuringiensis (Bt) toxins which are widely used for the biological control of insect pests and disease vectors. Bt toxins, after solubilization and activation by midgut proteases, bind to specific high-affinity receptors (aminopeptidases N, cadherins and possibly other proteins) at the surface of the brush-border apical membrane of epithelial cells. The toxins insert then into the membrane to form ionic pores (Schnepf et al., 1998). The subsequent disruption of ionic and osmotic gradients leads to cell lysis and eventually insect death by either infection or starvation (Knowles and Ellar, 1987). Recent investigations on Bt toxins focused on the elucidation of their structure–function relationships and the mechanisms of protein insertion and pore formation, and relied mainly on three experimental models: insect cell lines, midgut brush-border membrane vesicles (BBMVs) and artificial planar lipid bilayers (Schwartz and Laprade, 2000). Therefore, the availability of physiologically competent models derived from the intact insect midgut epithelial layer should provide a crucial tool for the study of the mode of action of microbial pathogens and natural toxins. A better understanding of the mechanism of action of these agents should in turn allow us to develop new insecticides and design strategies to delay the development of insect resistance to pathogens.
The patch-clamp technique is a powerful electrophysiological approach used to obtain precise information about the properties and distribution of ion channels in living tissues and cells (Neher and Sakmann, 1976, Hamill et al., 1981). It was successfully applied to the apical and basal membranes of epithelial cells of various mammalian tissues and of insect Malpighian tubules (Nicolson and Isaacson, 1990, O’Donnell et al., 1998, O’Connor and Beyenbach, 2001), but was difficult to use on insect midgut cells, either in culture or in the intact midgut. The presence of Ba2+-sensitive cationic channels in patch-clamped basal membranes of freshly dissociated goblet cells was briefly reported (Moffett and Lewis, 1990). On the other hand, there was no direct evidence of ion channels in the apical membrane of columnar cells, but experiments using planar lipid bilayers, in which lepidopteran midgut BBMVs were incorporated, strongly suggested the presence of various channel types, most of them being slightly selective to cations (Lorence et al., 1995, Peyronnet et al., 2000).
In this study, we adapted our intact larval midgut preparation (Peyronnet et al., 1997) to the patch-clamp technique. Using this model, we provided the first direct evidence for the existence of endogenous ion channels in the apical membrane of epithelial cells from freshly isolated Manduca sexta larval midguts. Microelectrode measurements were also performed on the epithelial cells of this preparation in order to investigate how their intracellular potential was related to the physiological condition of the larvae. Finally, the patch-clamp technique was used to explore the interaction between the trypsin-activated Cry1Ac toxin, a Bt pore-forming toxin, and the apical membrane of the epithelial cells of M. sexta larval midguts.
Section snippets
Solutions
Midgut dissection and electrophysiological experiments were conducted in a buffered standard 32K solution modified from Moffett and Koch (1988) and composed of 32 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 166 mM sucrose and 5 mM Tris–HCl (pH 9.0). In patch-clamp experiments, this solution was used in both bath and pipette, except for ionic selectivity studies where the bath was perfused with a solution containing 100 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 30 mM sucrose and 5 mM Tris–HCl (pH 9.0). Solutions were
Microelectrode experiments
Impalements were successful in about 96% of the midgut preparations (n=56). Because goblet cells are recessed and less numerous in larval midguts, intracellular potentials were most probably made on columnar cells. Vi ranged from −33.7 to −85.5 mV, depending on the physiological state of the larvae. In each midgut preparation, it did not differ by more than a few millivolts from cell to cell and remained usually stable for at least 30 min. Vi from actively feeding third-instar larvae (stage A)
Discussion
In the present study, the patch-clamp technique was used, for the first time, to explore ion channel activity in the apical membrane of insect midgut cells. Although numerous reports have been published on potassium ion transport pathways in the insect midgut and particularly in midgut goblet cells (reviewed in Klein et al., 1996, Gringorten, 2001), little information is available on the movement of ions across the apical membrane of columnar cells, with the exception of K+/amino acid
Acknowledgements
We are grateful to Lucie Marceau and Marc Juteau (Biocontrol Network and GÉPROM, Université de Montréal) for preparing the Cry1Ac toxin, to Mireille Marsolais (Biocontrol Network and GÉPROM, Université de Montréal), Geneviève Larouche, Benoît Rancourt and Charles Vincent (Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada) for their assistance in rearing the insects and to Vincent Vachon (Biocontrol Network and GÉPROM,
References (54)
- et al.
Establishment of primary and continuous cultures of epithelial cells from larval lepidopteran midguts
Journal of Insect Physiology
(1994) - et al.
Growth and differentiation of the larval midgut epithelium during molting in the moth, Manduca sexta
Tissue and Cell
(1991) - et al.
The amino acid/K+ symporters for neutral amino acids along the midgut of lepidopteran larvae: functional differentiations
Journal of Insect Physiology
(1994) - et al.
Purification and properties of a cytosolic V1-ATPase
Journal of Biological Chemistry
(1996) - et al.
Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta
Tissue and Cell
(1988) - et al.
Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different specificity
Biochimica et Biophysica Acta
(1987) - et al.
δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers
Federation of European Biochemical Society Letters
(1995) - et al.
Expression of a midgut-specific cadherin BT-R1 during the development of Manduca sexta larva
Comparative Biochemistry and Physiology Part B
(2003) - et al.
Patch clamp of the basal membrane of beetle Malpighian tubules: direct demonstration of potassium channels
Journal of Insect Physiology
(1990) - et al.
A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb+–K+ efflux in vitro
Journal of Biological Chemistry
(1994)
Early response of cultured lepidopteran cells to exposure to δ-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels
Biochimica et Biophysica Acta
Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits
Journal of Biological Chemistry
A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut
Journal of Biological Chemistry
A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane
Journal of Biological Chemistry
Localization of amino acid absorption systems in the larval midgut of the tobacco hornworm Manduca sexta
Journal of Insect Physiology
Discrete localisation of distinct alkaline phosphatase isoenzymes in the cell surface of silkworm midgut epithelium
Journal of Experimental Zoology
Midgut development
Structure and ultrastructure of the insect midgut
Ion transport across the midgut of the tobacco hornworm (Manduca sexta)
Journal of Experimental Biology
Changes in midgut active ion transport and metabolism during the fifth instar of the tobacco hornworm (Manduca sexta)
Journal of Experimental Zoology
Changes in midgut active ion transport and metabolism during larval–larval molting in the tobacco hornworm (Manduca sexta)
Journal of Experimental Biology
Comparison of potassium transport in three structurally distinct regions of the insect midgut
Journal of Experimental Biology
Role of midgut electrogenic K+ pump potential difference in regulating lumen K+ and pH in larval Lepidoptera
Journal of Experimental Biology
Midgut metabolism in different instars of the tobacco hornworm (Manduca sexta)
Journal of Experimental Zoology
Amino acid transport systems in intestinal brush-border membranes from lepidopteran larvae
American Journal of Physiology
Ion balance in the lepidopteran midgut and insecticidal action of Bacillus thuringiensis
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
Pflügers Archives
Cited by (6)
The paracellular pathway in the lepidopteran larval midgut: Modulation by intracellular mediators
2006, Comparative Biochemistry and Physiology - A Molecular and Integrative PhysiologyCitation Excerpt :The electrogenic activity of the proton pump and the high luminal K+ concentration provide the electrochemical gradient for K+ influx across the apical membrane of columnar cells that drives amino acid accumulation by means of specific K+/amino acid symporters (Giordana et al., 1998; Castagna et al., 1998; Casartelli et al., 2001). Therefore, apical K+ channels in columnar cells are not expressed or are inactive in feeding larvae (Peyronnet et al., 2004), a channel activity becoming evident only in moulting or starved larvae, or in larvae exposed to particular environmental conditions. Cl− permeability across the columnar cell apical membrane is exclusively due to a Cl−/HCO3− exchanger (Chao et al., 1989).
Canadian contributions to forest insect pathology and to the use of pathogens in forest pest management
2016, Canadian EntomologistEfficiency and midgut histopathological effect of the newly isolated Bacillus thuringiensis KS δ-Endotoxins on the emergent pest Tuta absoluta
2013, Journal of Microbiology and Biotechnology
- 1
These authors contributed equally to this work.