CL- CHANNELS IN BASOLATERAL RENAL MEDULLARY MEMBRANES .7. CHARACTERIZATION OF THE INTRACELLULAR ANION-BINDING SITES

A unique property of basolateral membrane Cl- channels from the mTAL i s that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P(o)). Th e K1/2 for maximal activation of P(o) by Cl- facing intracellular doma ins of these channels is 10 mm Cl-. The present experiments evaluated the nature of these Cl--interactive sites. First, we found that the im permeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment P(o) with a K1/2 in the range of 10 mm isethionat e without affecting conductance (g(Cl), pS). Second, pretreatment of t he solutions facing the intracellular aspects of the channels with eit her 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lys ine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P(o) usually seen when the Cl- concentrat ion of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl- channel activity was increased by fi rst raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solution s bathing intracellular Cl- channel faces had no effect on P(o). We co nclude that the intracellular aspects of these Cl- channels contain Cl --interactive loci (termed [Cl](i)) which are accessible to impermeant anions in intracellular fluids and which contain arginine- and lysine -rich domains which can be inactivated, at low ambient Cl- or isethion ate concentrations, by interactions with PGO or TNBS.