Cl^- channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

A unique property of basolateral membrane Cl^- channels from the mTAL is that the Cl^- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P₀). The K_1/2 for maximal activation of P₀ by Cl^- facing intracellular domains of these channels is 10 m m Cl^-. The present experiments evaluated the nature of these Cl^--interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl^- channel faces, could augment P₀ with a K_1/2 in the range of 10 m m isethionate without affecting conductance (g_Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 m m phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 m m), prevented the activation of P₀ usually seen when the Cl^- concentration of solutions facing intracellular channel domains was raised from 2 to 50 m m. However, when the Cl^- channel activity was increased by first raising the Cl^- concentration bathing intracellular channel faces from 2 to 50 m m, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl^- channel faces had no effect on P₀. We conclude 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 arginineand lysine-rich domains which can be inactivated, at low ambient Cl^- or isethionate concentrations, by interactions with PGO or TNBS.