Evidence for a cytosolic inhibitor of epithelial chloride channels

Abstract

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Characterization of Cytosolic Cl^– Channel Inhibitors by Size Exclusion Chromatography

“…As shown earlier, this procedure leaves the cytosolic in hibitors unaffected [17][18][19]. To enrich these inhibitors the filtrate containing heat-stable solutes was fractionated by size exclusion high-performance liquid chromatography (HPLC) using a Superose 12 column.…”

“…The experiments were performed on subcontluent monolayer cultures of HT29 cells grown on collagencoated cover slips as described previously [18,19] and bathed in NaCl-Ringer solution of plasma-like ion composition. Conventional patch-clamp techniques were used to measure single channel currents in ex cised inside-out patches at room temperature usually on the first or second day of cell seeding.…”

“…More over, we have subjected a number of putative inhibitors of CP channels to gel chromatogra phy column to compare their behaviour with that of the cytosolic inhibitor. Part of the work has been published in abstract form [20].…”

“…Other CP channels were inhibit ed by intra-or extracellular nucleotides and nucleotide analogs [11][12][13][14], Futhermore, cisunsaturated fatty acids, e.g. arachidonic acid, caused inhibition of CP channels [15][16][17], Recently, we have demonstrated that cyto sol from pig kidney, human placenta, HT29 and CFPAC-1 cells inhibits outwardly rectify ing CP channels from respiratory epithelial cells, CFPAC-1 cells, and from HT29 cells [17][18][19], The inhibition showed a rapid onset, was concentration-dependent, reversible, and was caused by a heat-stable factor of unknown structure. Here we report on the fractionation of the cytosol by gel chromatography as a first step to isolate the inhibitory factors.…”

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.…”