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 dosedependent 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°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 <5kDa (n=13) and probably <1.5 kDa (n=11), but >700 kDa (n=9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, α-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. CI is heat-stable and amphiphilic and has an apparent molecular mass of 0.7–1.5 kDa. Given this nature of CI, several putative ion-channel regulators were examined on Cl⁻ channels of HT₂₉ cells. It was found that inositol triphosphate, GTP, GTP [γ-S], ATP, cAMP, cGMP and dioleoylglycerol all had no effect from the cytosolic side. Non-saturated fatty acids (n=23) inhibited the open probability of these Cl⁻ channels from the cytosolic side after some delay reversibly at concentrations of 5 μmol/l for arachidonic acid and more than 1 mmol/l for linoleic acid. Saturated fatty acids had no effect. The present data indicate that this type of Cl⁻ channel may be inhibited by some cytosolic inhibitor with the above properties. Excision of membrane patches containing this channel leads to instantaneous disinhibition (=excision activation). It is possible that an increased concentration of CI or an increased sensitivity to CI may be responsible for the “tonic inhibition” of Cl⁻ channels observed in cystic fibrosis REC.