hide
Free keywords:
Renal brush-border membrane; Cl− conductance; Outwardly rectifying Cl− channel; Cytosol preparation; Heat-stable cytosolic Cl− channel inhibitor
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 Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl− conductance in the brush-border membrane, which is activated during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.
