Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 May;80(5):2198–2206. doi: 10.1016/S0006-3495(01)76192-9

Chemically charging the pore constriction opens the mechanosensitive channel MscL.

K Yoshimura 1, A Batiza 1, C Kung 1
PMCID: PMC1301411  PMID: 11325722

Abstract

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.

Full Text

The Full Text of this article is available as a PDF (108.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ajouz B., Berrier C., Garrigues A., Besnard M., Ghazi A. Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells. J Biol Chem. 1998 Oct 9;273(41):26670–26674. doi: 10.1074/jbc.273.41.26670. [DOI] [PubMed] [Google Scholar]
  2. Akabas M. H., Stauffer D. A., Xu M., Karlin A. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science. 1992 Oct 9;258(5080):307–310. doi: 10.1126/science.1384130. [DOI] [PubMed] [Google Scholar]
  3. Bargmann C. I. Molecular mechanisms of mechanosensation? Cell. 1994 Sep 9;78(5):729–731. doi: 10.1016/s0092-8674(94)90382-4. [DOI] [PubMed] [Google Scholar]
  4. Berrier C., Besnard M., Ajouz B., Coulombe A., Ghazi A. Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J Membr Biol. 1996 May;151(2):175–187. doi: 10.1007/s002329900068. [DOI] [PubMed] [Google Scholar]
  5. Berrier C., Garrigues A., Richarme G., Ghazi A. Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel mscL. J Bacteriol. 2000 Jan;182(1):248–251. doi: 10.1128/jb.182.1.248-251.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bloomfield V. Editorial. Biophys J. 1993 Jul;65(1):1–1. doi: 10.1016/S0006-3495(93)81020-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blount P., Schroeder M. J., Kung C. Mutations in a bacterial mechanosensitive channel change the cellular response to osmotic stress. J Biol Chem. 1997 Dec 19;272(51):32150–32157. doi: 10.1074/jbc.272.51.32150. [DOI] [PubMed] [Google Scholar]
  8. Blount P., Sukharev S. I., Moe P. C., Martinac B., Kung C. Mechanosensitive channels of bacteria. Methods Enzymol. 1999;294:458–482. doi: 10.1016/s0076-6879(99)94027-2. [DOI] [PubMed] [Google Scholar]
  9. Blount P., Sukharev S. I., Schroeder M. J., Nagle S. K., Kung C. Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11652–11657. doi: 10.1073/pnas.93.21.11652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang G., Spencer R. H., Lee A. T., Barclay M. T., Rees D. C. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science. 1998 Dec 18;282(5397):2220–2226. doi: 10.1126/science.282.5397.2220. [DOI] [PubMed] [Google Scholar]
  11. Cruickshank C. C., Minchin R. F., Le Dain A. C., Martinac B. Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. Biophys J. 1997 Oct;73(4):1925–1931. doi: 10.1016/S0006-3495(97)78223-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. French A. S. Mechanotransduction. Annu Rev Physiol. 1992;54:135–152. doi: 10.1146/annurev.ph.54.030192.001031. [DOI] [PubMed] [Google Scholar]
  13. Hamill O. P., McBride D. W., Jr The pharmacology of mechanogated membrane ion channels. Pharmacol Rev. 1996 Jun;48(2):231–252. [PubMed] [Google Scholar]
  14. Karlin A., Akabas M. H. Substituted-cysteine accessibility method. Methods Enzymol. 1998;293:123–145. doi: 10.1016/s0076-6879(98)93011-7. [DOI] [PubMed] [Google Scholar]
  15. Larsson H. P., Baker O. S., Dhillon D. S., Isacoff E. Y. Transmembrane movement of the shaker K+ channel S4. Neuron. 1996 Feb;16(2):387–397. doi: 10.1016/s0896-6273(00)80056-2. [DOI] [PubMed] [Google Scholar]
  16. Levina N., Tötemeyer S., Stokes N. R., Louis P., Jones M. A., Booth I. R. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 1999 Apr 1;18(7):1730–1737. doi: 10.1093/emboj/18.7.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu Y., Holmgren M., Jurman M. E., Yellen G. Gated access to the pore of a voltage-dependent K+ channel. Neuron. 1997 Jul;19(1):175–184. doi: 10.1016/s0896-6273(00)80357-8. [DOI] [PubMed] [Google Scholar]
  18. Lu T., Nguyen B., Zhang X., Yang J. Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification. Neuron. 1999 Mar;22(3):571–580. doi: 10.1016/s0896-6273(00)80711-4. [DOI] [PubMed] [Google Scholar]
  19. Martinac B., Buechner M., Delcour A. H., Adler J., Kung C. Pressure-sensitive ion channel in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2297–2301. doi: 10.1073/pnas.84.8.2297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moe P. C., Levin G., Blount P. Correlating a protein structure with function of a bacterial mechanosensitive channel. J Biol Chem. 2000 Oct 6;275(40):31121–31127. doi: 10.1074/jbc.M002971200. [DOI] [PubMed] [Google Scholar]
  21. Nakamaru Y., Takahashi Y., Unemoto T., Nakamura T. Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock. FEBS Lett. 1999 Feb 12;444(2-3):170–172. doi: 10.1016/s0014-5793(99)00054-x. [DOI] [PubMed] [Google Scholar]
  22. Ou X., Blount P., Hoffman R. J., Kung C. One face of a transmembrane helix is crucial in mechanosensitive channel gating. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11471–11475. doi: 10.1073/pnas.95.19.11471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pascual J. M., Karlin A. State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit. J Gen Physiol. 1998 Jun;111(6):717–739. doi: 10.1085/jgp.111.6.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pascual J. M., Shieh C. C., Kirsch G. E., Brown A. M. K+ pore structure revealed by reporter cysteines at inner and outer surfaces. Neuron. 1995 May;14(5):1055–1063. doi: 10.1016/0896-6273(95)90344-5. [DOI] [PubMed] [Google Scholar]
  25. Sachs F., Morris C. E. Mechanosensitive ion channels in nonspecialized cells. Rev Physiol Biochem Pharmacol. 1998;132:1–77. doi: 10.1007/BFb0004985. [DOI] [PubMed] [Google Scholar]
  26. Sackin H. Mechanosensitive channels. Annu Rev Physiol. 1995;57:333–353. doi: 10.1146/annurev.ph.57.030195.002001. [DOI] [PubMed] [Google Scholar]
  27. Stauffer D. A., Karlin A. Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry. 1994 Jun 7;33(22):6840–6849. doi: 10.1021/bi00188a013. [DOI] [PubMed] [Google Scholar]
  28. Sukharev S. I., Blount P., Martinac B., Blattner F. R., Kung C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature. 1994 Mar 17;368(6468):265–268. doi: 10.1038/368265a0. [DOI] [PubMed] [Google Scholar]
  29. Sukharev S. I., Blount P., Martinac B., Kung C. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities. Annu Rev Physiol. 1997;59:633–657. doi: 10.1146/annurev.physiol.59.1.633. [DOI] [PubMed] [Google Scholar]
  30. Sukharev S. I., Schroeder M. J., McCaslin D. R. Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study. J Membr Biol. 1999 Oct 1;171(3):183–193. doi: 10.1007/s002329900570. [DOI] [PubMed] [Google Scholar]
  31. Sukharev S. I., Sigurdson W. J., Kung C., Sachs F. Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J Gen Physiol. 1999 Apr;113(4):525–540. doi: 10.1085/jgp.113.4.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wilson G. G., Karlin A. The location of the gate in the acetylcholine receptor channel. Neuron. 1998 Jun;20(6):1269–1281. doi: 10.1016/s0896-6273(00)80506-1. [DOI] [PubMed] [Google Scholar]
  33. Yang N., George A. L., Jr, Horn R. Molecular basis of charge movement in voltage-gated sodium channels. Neuron. 1996 Jan;16(1):113–122. doi: 10.1016/s0896-6273(00)80028-8. [DOI] [PubMed] [Google Scholar]
  34. Yang N., Horn R. Evidence for voltage-dependent S4 movement in sodium channels. Neuron. 1995 Jul;15(1):213–218. doi: 10.1016/0896-6273(95)90078-0. [DOI] [PubMed] [Google Scholar]
  35. Yoshimura K., Batiza A., Schroeder M., Blount P., Kung C. Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys J. 1999 Oct;77(4):1960–1972. doi: 10.1016/S0006-3495(99)77037-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang H., Karlin A. Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor. Biochemistry. 1998 Jun 2;37(22):7952–7964. doi: 10.1021/bi980143m. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES