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

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Abstract

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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References

  1. 1.

    Baylisse, J.M., Reeves, W.B., Andreoli, T.E. 1990. Cl transport in basolateral renal medullary vesicles. I. Cl transport in intact vesicles. J. Membrane Biol. 113:49–56

  2. 2.

    Freedman, R.B., Radda, G.K. 1968. The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids. Biochem. J. 108:383–391

  3. 3.

    Garty, H., Yeger, O., Asher, C. 1988. Sodium-dependent inhibition of the epithelial sodium channel by an arginylspecific reagent. J. Biol. Chem. 263:5550–5554

  4. 4.

    Greger, R., Oberleithner, H., Schlatter, E., Cassola, A.C., Weidtke, C. 1983. Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney. Pfluegers Arch. 399:20–34

  5. 5.

    Hebert, S.C., Andreoli, T.E. 1984. Effects of antidiuretic hormone on cellular conductance pathways in mouse medullary thick ascending limbs of Henle. II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl absorption. J. Membrane Biol. 80:221–233

  6. 6.

    Jentsch, T.J., Steinmeyer, K., Schwarz, G. 1990. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348:510–514

  7. 7.

    Kremer, A.B., Egan, R.M., Sable, H.Z. 1980. The active site of transketolase. Two arginine residues are essential for activity. J. Biol. Chem. 255:2405–2410

  8. 8.

    Lundblad, R.L. 1991. Chemical Reagents for Protein Modification. CRC, Boca Raton, FL

  9. 9.

    Molony, D.A., Andreoli, T.E. 1988. Diluting power of thick limbs of Henle. I. Peritubular hypertonicity blocks basolateral Cl channels. Am. J. Physiol. 255:F1128-F1137

  10. 10.

    Molony, D.A., Reeves, W.B., Hebert, S.C., Andreoli, T.E. 1987. ADH increases apical Na+:K+:2Cl entry in mouse medullary thick ascending limbs of Henle. Am. J. Physiol. 252:F177-F187

  11. 11.

    Reeves, W.B., Andreoli, T.E. 1990. Cl transport in basolateral renal medullary vesicles. II. Cl channels in planar lipid bilayers. J. Membrane Biol. 113:57–65

  12. 12.

    Schlatter, E., Greger, R. 1985. cAMP increases the basolateral Cl conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse. Pfluegers Arch. 405:367–376

  13. 13.

    Spires, S., Begenisich, T. 1992. Modification of potassium channel kinetics by amino group reagents. J. Gen. Physiol. 99:109–129

  14. 14.

    Steinmeyer, K., Ortland, C., Jentsch, T.J. 1991. Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel. Nature 354:301–304

  15. 15.

    Takahashi, K. 1968. The reaction of phenylglyoxal with arginine residues in proteins. J. Biol. Chem. 243:6171–6179

  16. 16.

    Winters, C.J., Reeves, W.B., Andreoli, T.E. 1991. Cl channels in basolateral renal medullary membranes. III. Determinants of single-channel activity. J. Membrane Biol. 118: 269–278

  17. 17.

    Winters, C.J., Reeves, W.B., Andreoli, T.E. 1991. Cl channels in basolateral renal medullary membrane vesicles. IV. Analogous channel activation by Cl or cAMP-dependent protein kinase. J. Membrane Biol. 122:89–95

  18. 18.

    Winters, C.J., Reeves, W.B., Andreoli, T.E. 1992. Cl channels in basolateral renal medullary vesicles. V. Comparison of basolateral mTALH Cl channels with apical Cl channels from jejunum and trachea. J. Membrane Biol. 128:27–39

  19. 19.

    Winters, C.J., Reeves, W.B., Andreoli, T.E. 1992. Intracellular anions modulate basolateral Cl channels by interacting with lysine or arginine-rich domains. J. Am. Soc. Nephrol. 3:823 (Abstr.)

  20. 20.

    Zimniak, L., Reeves, W.B., Andreoli, T.E. 1992. Cl channels in basolateral renal medullary membranes: VI. Expression of a chloride conductance in X. laevis oocytes. Am. J. Physiol. 263:F979-F984

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Additional information

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

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Key words

  • Cl channels
  • Cl-interactive loci
  • PGO
  • TNBS
  • Anion binding sites