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 (P 0 ). The K 1/2 for maximal activation of P 0 by Cl− facing intracellular domains of these channels is 10 mm 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 P 0 with a K 1/2 in the range of 10 mm isethionate without affecting conductance (g Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P 0 usually seen when the Cl− concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl− channel activity was increased by first raising the Cl− concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl− channel faces had no effect on P 0 . 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.
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We acknoeledge the able technical assistance of Anna Grace Stewart. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veteterans Administration Merit Review Grants to T. E.Andreoli and to W. B. Reeves. C. J. Winters is a Veterans Administration Associate Investigator.
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Winters, C.J., Reeves, W.B. & Andreoli, T.E. Cl− channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites. J. Membarin Biol. 135, 145–152 (1993). https://doi.org/10.1007/BF00231440
- Cl− channels
- Cl−-interactive loci
- Anion binding sites