Excretion

Two families of chloride channels in apical membrane patches of stellate cells of malpighian tubules of the yellow fever mosquito Aedes aegypti

K. O'Connor & K. W. Beyenbach

Dept. of Biomedical Sciences, Cornell University, VRT 8014, Ithaca, NY 14853, USA

Previous studies have shown that during leucokinin-VIII-stimulated diuresis, Cl - moves from the hemolymph to the tubule lumen either through paracellular septate junctions in Aedes Malpighian tubules (Pannabecker et al., J. Membr. Biol. 132, 63, 1993) or through stellate cells in Drosophila Malpighian tubules (O'Donnell et al., Am. J. Physiol. 274, R609, 1998). The apical membrane of Malpighian tubules of the yellow fever mosquito, Aedes aegypti, was exposed by hand-dissection, and patches of stellate cells were studied in the excised, inside-out configuration. The most frequently observed Cl - channel has a conductance of 27 pS and a high open probability and dwell time. This channel is blocked by DPC, 9-AC and niflumic acid from the intracellular (bath) side. SITS and DIDS were not effective blockers. Multiple channels of this type were often observed in the same patch (up to five in pipettes with 10-20 M? resistance), suggesting high channel density. The second Cl - channel has a conductance of 5 pS, a low open probability and dwell time and a very high selectivity for chloride. Leucokinin-VIII is thought to open the chloride pathway via Ca 2+ serving as a second messenger (Cady & Hagedorn, J. Insect Phsyiol. 45, 327, 1999). For this reason, bath Ca 2+ was eliminated. Yet this maneuver did not appear to have an effect on Cl - channel activity. Since these channels were highly active in excised patches under control conditions, it is possible that excising the membrane dissociates the channels from their regulatory constituents. Hence it may not be possible to directly link these Cl - channels to leucokinin-VIII-induced Cl - secretion using excised membrane patches. Finding a high density of Cl - channels in apical membrane patches of stellate cells in Aedes aegypti supports the O'Donnell view of transcellular Cl - movement during leucokinin-induced diuresis (Am. J. Physiol. 274, R1039, 1998). But stellate cells must also have Cl - channels in the basolateral membrane in order to explain transepithelial Cl - diffusion potentials that are symmetrical for lumen-to-bath and bath-to-lumen Cl - gradients. In addition, Cl - concentrations in the cytoplasm must rise and fall with extracellular Cl - concentration in order to explain a shunt conductance that is dependent on the Cl - concentration in both luminal and peritubular fluids (Pannabecker et al., J. Membr. Biol. 132, 63, 1993). Thus, it is unlikely, but not impossible, that stellate cells serve as the Cl - shunt pathway under conditions of diuresis induced by leucokinin. (NSF IBN 9604394)

Index terms: leucokinin, stellate cells, calcium, shunt pathway, septate junctions.

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