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. 2001 Apr;80(4):1810–1818. doi: 10.1016/S0006-3495(01)76151-6

Covalently linked gramicidin channels: effects of linker hydrophobicity and alkaline metals on different stereoisomers.

K M Armstrong 1, E P Quigley 1, P Quigley 1, D S Crumrine 1, S Cukierman 1
PMCID: PMC1301370  PMID: 11259294

Abstract

The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs(+) or K(+)) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs(+)- or K(+)-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.

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Selected References

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  1. Andersen O. S., Apell H. J., Bamberg E., Busath D. D., Koeppe R. E., 2nd, Sigworth F. J., Szabo G., Urry D. W., Woolley A. Gramicidin channel controversy--the structure in a lipid environment. Nat Struct Biol. 1999 Jul;6(7):609–612. doi: 10.1038/10648. [DOI] [PubMed] [Google Scholar]
  2. Arseniev A. S., Barsukov I. L., Bystrov V. F., Lomize A. L., Ovchinnikov YuA 1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices. FEBS Lett. 1985 Jul 8;186(2):168–174. doi: 10.1016/0014-5793(85)80702-x. [DOI] [PubMed] [Google Scholar]
  3. Bamberg E., Janko K. The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes. Biochim Biophys Acta. 1977 Mar 17;465(3):486–499. doi: 10.1016/0005-2736(77)90267-x. [DOI] [PubMed] [Google Scholar]
  4. Burkhart B. M., Li N., Langs D. A., Pangborn W. A., Duax W. L. The conducting form of gramicidin A is a right-handed double-stranded double helix. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12950–12955. doi: 10.1073/pnas.95.22.12950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burkhart BM, Duax WL. Gramicidin channel controversy - reply. Nat Struct Biol. 1999 Jul;6(7):611–612. doi: 10.1038/10652. [DOI] [PubMed] [Google Scholar]
  6. Busath D. D. The use of physical methods in determining gramicidin channel structure and function. Annu Rev Physiol. 1993;55:473–501. doi: 10.1146/annurev.ph.55.030193.002353. [DOI] [PubMed] [Google Scholar]
  7. Chiu S. W., Subramaniam S., Jakobsson E. Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex. Biophys J. 1999 Apr;76(4):1929–1938. doi: 10.1016/S0006-3495(99)77352-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross T. A., Arseniev A., Cornell B. A., Davis J. H., Killian J. A., Koeppe R. E., 2nd, Nicholson L. K., Separovic F., Wallace B. A. Gramicidin channel controversy--revisited. Nat Struct Biol. 1999 Jul;6(7):610–612. doi: 10.1038/10650. [DOI] [PubMed] [Google Scholar]
  9. Cross T. A. Solid-state nuclear magnetic resonance characterization of gramicidin channel structure. Methods Enzymol. 1997;289:672–696. doi: 10.1016/s0076-6879(97)89070-2. [DOI] [PubMed] [Google Scholar]
  10. Crouzy S., Woolf T. B., Roux B. A molecular dynamics study of gating in dioxolane-linked gramicidin A channels. Biophys J. 1994 Oct;67(4):1370–1386. doi: 10.1016/S0006-3495(94)80618-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cukierman S. Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels. Biophys J. 2000 Apr;78(4):1825–1834. doi: 10.1016/S0006-3495(00)76732-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cukierman S., Quigley E. P., Crumrine D. S. Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers. Biophys J. 1997 Nov;73(5):2489–2502. doi: 10.1016/S0006-3495(97)78277-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duca K. A., Jordan P. C. Ion-water and water-water interactions in a gramicidinlike channel: effects due to group polarizability and backbone flexibility. Biophys Chem. 1997 Apr 22;65(2-3):123–141. doi: 10.1016/s0301-4622(96)02233-8. [DOI] [PubMed] [Google Scholar]
  14. Ketchem R. R., Hu W., Cross T. A. High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science. 1993 Sep 10;261(5127):1457–1460. doi: 10.1126/science.7690158. [DOI] [PubMed] [Google Scholar]
  15. Ketchem R., Roux B., Cross T. High-resolution polypeptide structure in a lamellar phase lipid environment from solid state NMR derived orientational constraints. Structure. 1997 Dec 15;5(12):1655–1669. doi: 10.1016/s0969-2126(97)00312-2. [DOI] [PubMed] [Google Scholar]
  16. Koeppe R. E., 2nd, Anderson O. S. Engineering the gramicidin channel. Annu Rev Biophys Biomol Struct. 1996;25:231–258. doi: 10.1146/annurev.bb.25.060196.001311. [DOI] [PubMed] [Google Scholar]
  17. Kolb H. A., Bamberg E. Influence of membrane thickness and ion concentration on the properties of the gramicidin a channel. Autocorrelation, spectral power density, relaxation and single-channel studies. Biochim Biophys Acta. 1977 Jan 4;464(1):127–141. doi: 10.1016/0005-2736(77)90376-5. [DOI] [PubMed] [Google Scholar]
  18. Lee W. K., Jordan P. C. Molecular dynamics simulation of cation motion in water-filled gramicidinlike pores. Biophys J. 1984 Dec;46(6):805–819. doi: 10.1016/S0006-3495(84)84079-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lundbaek J. A., Maer A. M., Andersen O. S. Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels. Biochemistry. 1997 May 13;36(19):5695–5701. doi: 10.1021/bi9619841. [DOI] [PubMed] [Google Scholar]
  20. Nagle J. F., Morowitz H. J. Molecular mechanisms for proton transport in membranes. Proc Natl Acad Sci U S A. 1978 Jan;75(1):298–302. doi: 10.1073/pnas.75.1.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pomès R., Roux B. Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. Biophys J. 1996 Jul;71(1):19–39. doi: 10.1016/S0006-3495(96)79211-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Quigley E. P., Crumrine D. S., Cukierman S. Gating and permeation in ion channels formed by gramicidin A and its dioxolane-linked dimer in Na(+) and Cs(+) solutions. J Membr Biol. 2000 Apr 1;174(3):207–212. doi: 10.1007/s002320001045. [DOI] [PubMed] [Google Scholar]
  23. Quigley E. P., Emerick A. J., Crumrine D. S., Cukierman S. Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers. Biophys J. 1998 Dec;75(6):2811–2820. doi: 10.1016/S0006-3495(98)77724-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Quigley E. P., Quigley P., Crumrine D. S., Cukierman S. The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels. Biophys J. 1999 Nov;77(5):2479–2491. doi: 10.1016/S0006-3495(99)77084-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ring A., Sandblom J. Modulation of gramicidin A open channel lifetime by ion occupancy. Biophys J. 1988 Apr;53(4):549–559. doi: 10.1016/S0006-3495(88)83135-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roux B., Karplus M. Ion transport in a model gramicidin channel. Structure and thermodynamics. Biophys J. 1991 May;59(5):961–981. doi: 10.1016/S0006-3495(91)82311-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. SARGES R., WITKOP B. GRAMICIDIN A. V. THE STRUCTURE OF VALINE- AND ISOLEUCINE-GRAMICIDIN A. J Am Chem Soc. 1965 May 5;87:2011–2020. doi: 10.1021/ja01087a027. [DOI] [PubMed] [Google Scholar]
  28. Stankovic C. J., Heinemann S. H., Delfino J. M., Sigworth F. J., Schreiber S. L. Transmembrane channels based on tartaric acid-gramicidin A hybrids. Science. 1989 May 19;244(4906):813–817. doi: 10.1126/science.2471263. [DOI] [PubMed] [Google Scholar]
  29. Urry D. W., Goodall M. C., Glickson J. D., Mayers D. F. The gramicidin A transmembrane channel: characteristics of head-to-head dimerized (L,D) helices. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1907–1911. doi: 10.1073/pnas.68.8.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Urry D. W. The gramicidin A transmembrane channel: a proposed pi(L,D) helix. Proc Natl Acad Sci U S A. 1971 Mar;68(3):672–676. doi: 10.1073/pnas.68.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wallace BA. Recent Advances in the High Resolution Structures of Bacterial Channels: Gramicidin A. J Struct Biol. 1998;121(2):123–141. doi: 10.1006/jsbi.1997.3948. [DOI] [PubMed] [Google Scholar]
  32. Woolf T. B., Roux B. Structure, energetics, and dynamics of lipid-protein interactions: A molecular dynamics study of the gramicidin A channel in a DMPC bilayer. Proteins. 1996 Jan;24(1):92–114. doi: 10.1002/(SICI)1097-0134(199601)24:1<92::AID-PROT7>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]

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