DPC blockade of transepithelial chloride absorption and single anion channels in teleost urinary bladder

Abstract

The columnar cell epithelium of the euryhaline goby (Gillichthys mirabilis) urinary bladder actively absorbs NaCl from the lumen, thereby driving water transport and reducing water loss to the hypertonic external environment. Transcellular transport of Cl- involves apical membrane entry via Na(+)-coupled cotransport driven by the Na+ electrochemical gradient and subsequent basolateral membrane exit. An anion channel in the basolateral cell membrane of columnar epithelial cells was identified using patch-clamp technique. This channel may be one avenue for basolateral Cl- exit from the urinary bladder columnar cell. Single-channel conductance (Gc) of channels in excised, inside-out membrane patches was approximately 75 pS in symmetrical solutions containing 140 mM Cl-. The channel was selective to Cl- over other anions [Cl- > 2-(N-morpholino)ethanesulfonic acid (MES) > F- approximately Br- approximately I- > NO3- approximately SO4(2-)). Channel activity, expressed as the open probability (Po), was voltage dependent in the physiological range of membrane potential, with membrane depolarization increasing Po. Decreasing the pH of the solution bathing the cytoplasmic face of the membrane patch over the range 8.4-6.0 reduced Po. There was no effect of pH on either Gc or ionic selectivity. Radiochloride flux technique was also applied to intact columnar epithelial cell sheets to relate anion channel activity to macroscopic transcellular transport. Serosal exposure to the anion channel blocker diphenylamine-2-carboxylic acid (DPC, 30 microM to 3 mM) reduced and abolished transcellular radiochloride fluxes and net Cl- absorption across short-circuited tissues in a dose-dependent fashion. DPC addition (10 microM to 1 mM) to the solution bathing the cytoplasmic face of excised, inside-out membrane patches reduced Po in a dose-dependent manner and had no effect on Gc. These parallel findings of DPC blockade on intact epithelia and on single anion channels support the notion that this anion channel is a basolateral membrane component of the pathway for Cl- movement in transcellular Cl- absorption.