Inhibition of epithelial chloride channels by cytosol

Abstract

Chloride channels that have an intermediate conductance and are outwardly rectifying were studied by the patch-clamp technique in cell-excised membrane patches from respiratory epithelial cells in primary culture (REC) of normal and cystic fibrosis tissue, HT29 and T84 human colon carcinoma cells and placenta trophoblast cells (PTC). Chloride channels were immediately activated by the exposure of the cytosolic side of the patch to a Ringer-type solution, which lacked cytosolic components normally inhibiting chloride channels in the "on" cell configuration. Tentatively, we labelled the cytosolic component (or components) responsible for this inhibition cytosolic inhibitor (CI). The presence of CI in cytosol derived from HT29 cells was shown by assaying crude cytosol extracts from these cells on Cl- channels from HT29 cells (n = 2) and REC from normal subjects and cystic fibrosis patients (n = 4). In order to examine CI further, PTC were used as a source of cytosol. The cytosol of PTC inhibited HT29 Cl- channels in a dose-dependent manner with a half-maximal inhibition observed at a 1:6 dilution (n = 11) of the native cytosol. CI from PTC was heat-stable (10 min at 100 degrees C, n = 8). When cytosol extract was partitioned into a chloroform phase, Cl- channel inhibition was shown for the lipophilic extract (n = 12) as well as for the aqueous phase (n = 10). The inhibitory potency of the lipid extract was slightly larger than that of the aqueous phase. Several separation procedures were used to determine the molecular size of CI. When CI was filtered through 30-kDa filters at 6000 rpm for 45 min, inhibitory potency was observed in the filtrate and the retained fraction (n = 3). The same was observed with 10-kDa filters (n = 6). When CI was dialysed through a 12-kDa membrane, inhibitory capacity was recovered from the dialysate. Similarly, gel filtration indicated that CI was less than 5 kDa (n = 13) and probably less than 1.5 kDa (n = 11), but greater than 700 kDa (n = 9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, alpha-amylase, nucleotidase), but none of the enzymes used destroyed the inhibitory potency of CI. These data indicate that CI is present in HT29 as well as in PTC. It inhibits reversibly intermediate-conductance outwardly rectifying Cl- channels in REC, HT29, and PTC.(ABSTRACT TRUNCATED AT 400 WORDS)

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel

“…CFTR has been shown to regulate intracellular ceramide levels and to affect apoptosis by controlling glutathione transport. It is also entirely possible that the soluble cytosolic inhibitor of ORCC described earlier, is related to the metabolism of reactive oxygen species (17,27,28).…”

ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family

“…Third, C1C-6 and C1C-7 may be channels which are regulated in a complicated way which we have not yet understood. In fact, many patch-clamp studies have revealed chloride channels which can only be observed after patch-excision [33]. Fourth, these channels may reside in intracellular organelles, which are known to possess chloride channels as well [34][35][36].…”

Anion Channels in the Apical Membrane of Collecting Duct Principal Cells in Culture

“…The latter agrees with our present obser vations. On the other hand, it has been found that the outwardly rectifying Cl" channel can be activated by excising a membrane patch from its cell [5,19,33] which probably re flects detachment of a cytosolic inhibitor from the channel [ 18,20], This may also hold for the present experiments, since the rectify ing channel -in contrast to the bursting Cl" channel and the voltage-gated anion channel -was only observed in excised patches.…”