Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

  • 20 Accesses

  • 5 Citations

Abstract

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179–188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257–261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.

This is a preview of subscription content, log in to check access.

References

  1. 1.

    Lauf PK, Theg BE: A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and ghost erythrocytes. Biophys Biochem Res Commun 92: 1422–1428, 1980

  2. 2.

    Lauf PK: Thiol-dependent passive K/Cl transport in sheep red cells. II. Loss of Cl and N-ethylmaleimide sensitivity in maturing high K+ cells. J Memb Biol 73: 247–256, 1983

  3. 3.

    Lauf PK, Adragna NC, Garay RP: Activation by N-ethylmaleimide of a latent K+-Cl flux in human red blood cells. Am J Physiol 246: C385-C390, 1984

  4. 4.

    Logue P, Anderson C, Kanik C, Farquharson B, Dunham P: Passive potassium transport in LK sheep red cells. Modification by N-ethylmaleimide. J Gen Physiol 81: 761–885, 1983

  5. 5.

    Kaji D, Kahn T: Kinetics of Cl-dependent K influx in human erythrocytes with and without external Na: Effect of NEM. Am J Physiol 249: C490–0496, 1985

  6. 6.

    Wiater LA, Dunham PB: Passive transport of K+ and Na+ in human red blood cells: Sulfhydryl binding agents and furosemide. Am J Physiol 245: C348-C356, 1983

  7. 7.

    Bauer J, Lauf PK: Thiol-dependent passive K/Cl transport in sheep red cells: III. Differential reactivity of membrane SH groups with N-ethylmaleimide and iodoacetamide. J Memb Biol 73: 257–261, 1983

  8. 8.

    Lauf PK: Thiol-dependent passive K/Cl transport in sheep red cells: VII. Volume-independent freezing by iodoacetamide, and sulfhydryl group heterogeneity. J Memb Biol 98: 237–246, 1987

  9. 9.

    Lauf PK: Kinetic comparison of ouabain-resistant K:CI fluxes (K:CI (Co)-transport) stimulated in sheep erythrocytes by membrane thiol oxidation and alkylation. Molecular and Cellular Biochemistry 82: 97–106, 1988

  10. 10.

    Lauf PK: Thiol-dependent K:CI transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide. J Memb Biol 101: 179–188, 1988

  11. 11.

    Lauf PK, Theg BE: N-ethylmaleimide enhances selectively passive K+ permeability in low potassium sheep red cells. In: SR Hollán, G Gárdos and B Sarkadi (eds). Adv Physiol Sci Vol 6. Genetics. Structure and function of blood cells. Pergamon Press, Budapest, Hungary, 1980, pp 285–291

  12. 12.

    Ryu KH, Lauf PK: Characterization of inhibitory SH group (s) in the thiol activated K:Cl cotransporter of low K (LK) sheep red blood cells. (Abstr) Biophys J 55: 141a, 1989

  13. 13.

    Lauf PK: Thiol-dependent passive K/Cl transport in sheep red cells: I. Dependence on chloride and external K+[Rb+] ions. J Memb Biol 73: 237–246, 1983

  14. 14.

    Abbott RE, Schachter D: Impermeant Maleimides. Oriented probes of erythrocyte membrane proteins. J Biol Chem 251: 7176–7183, 1976

  15. 15.

    Lauf PK: Thiol-dependent passive K+Cl transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes. J Memb Biol 82: 167–178, 1984

  16. 16.

    Lauf PK: K+:Cl cotransport: sulfhydryls, divalent cations, and the mechanism of volume activation in a red cell. J Memb Biol 88: 1–13, 1985

  17. 17.

    Lauf PK: K:Cl cotransport: Emerging molecular aspects of a ouabain-resistant, volume-responsive transport system in red blood cells. Renal Physiol Biochem 3–5: 248–259, 1988

Download references

Author information

Correspondence to P. K. Lauf.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ryu, K., Lauf, P.K. Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells. Mol Cell Biochem 99, 135–140 (1990). https://doi.org/10.1007/BF00230343

Download citation

Key words

  • K:Cl cotransport
  • chemical activation
  • thiol groups
  • mono-bifunctional SH reagents
  • sheep erythrocytes