Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1993 Apr 1;101(4):545–569. doi: 10.1085/jgp.101.4.545

Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites

PMCID: PMC2216774  PMID: 8389396

Abstract

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6- trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP- ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Alton E. W., Manning S. D., Schlatter P. J., Geddes D. M., Williams A. J. Characterization of a Ca(2+)-dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers. J Physiol. 1991 Nov;443:137–159. doi: 10.1113/jphysiol.1991.sp018827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. P., Berger H. A., Rich D. P., Gregory R. J., Smith A. E., Welsh M. J. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell. 1991 Nov 15;67(4):775–784. doi: 10.1016/0092-8674(91)90072-7. [DOI] [PubMed] [Google Scholar]
  3. Ashcroft F. M. Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci. 1988;11:97–118. doi: 10.1146/annurev.ne.11.030188.000525. [DOI] [PubMed] [Google Scholar]
  4. Ashcroft F. M., Harrison D. E., Ashcroft S. J. Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. 1984 Nov 29-Dec 5Nature. 312(5993):446–448. doi: 10.1038/312446a0. [DOI] [PubMed] [Google Scholar]
  5. Bean B. P. ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics. J Neurosci. 1990 Jan;10(1):1–10. doi: 10.1523/JNEUROSCI.10-01-00001.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bean B. P. Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci. 1992 Mar;13(3):87–90. doi: 10.1016/0165-6147(92)90032-2. [DOI] [PubMed] [Google Scholar]
  7. Bridges R. J., Garty H., Benos D. J., Rummel W. Na+ uptake into colonic enterocyte membrane vesicles. Am J Physiol. 1988 Apr;254(4 Pt 1):C484–C490. doi: 10.1152/ajpcell.1988.254.4.C484. [DOI] [PubMed] [Google Scholar]
  8. Bridges R. J., Worrell R. T., Frizzell R. A., Benos D. J. Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers. Am J Physiol. 1989 Apr;256(4 Pt 1):C902–C912. doi: 10.1152/ajpcell.1989.256.4.C902. [DOI] [PubMed] [Google Scholar]
  9. Christine C. W., Choi D. W. Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons. J Neurosci. 1990 Jan;10(1):108–116. doi: 10.1523/JNEUROSCI.10-01-00108.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colquhoun D., Hawkes A. G. On the stochastic properties of bursts of single ion channel openings and of clusters of bursts. Philos Trans R Soc Lond B Biol Sci. 1982 Dec 24;300(1098):1–59. doi: 10.1098/rstb.1982.0156. [DOI] [PubMed] [Google Scholar]
  11. Colquhoun D., Ogden D. C. Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol. 1988 Jan;395:131–159. doi: 10.1113/jphysiol.1988.sp016912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coronado R., Miller C. Conduction and block by organic cations in a K+-selective channel from sarcoplasmic reticulum incorporated into planar phospholipid bilayers. J Gen Physiol. 1982 Apr;79(4):529–547. doi: 10.1085/jgp.79.4.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DEL CASTILLO J., KATZ B. Interaction at end-plate receptors between different choline derivatives. Proc R Soc Lond B Biol Sci. 1957 May 7;146(924):369–381. doi: 10.1098/rspb.1957.0018. [DOI] [PubMed] [Google Scholar]
  14. Egan M., Flotte T., Afione S., Solow R., Zeitlin P. L., Carter B. J., Guggino W. B. Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. Nature. 1992 Aug 13;358(6387):581–584. doi: 10.1038/358581a0. [DOI] [PubMed] [Google Scholar]
  15. Grubmeyer C., Penefsky H. S. Cooperatively between catalytic sites in the mechanism of action of beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1981 Apr 25;256(8):3728–3734. [PubMed] [Google Scholar]
  16. Grubmeyer C., Penefsky H. S. The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1981 Apr 25;256(8):3718–3727. [PubMed] [Google Scholar]
  17. Halm D. R., Frizzell R. A. Anion permeation in an apical membrane chloride channel of a secretory epithelial cell. J Gen Physiol. 1992 Mar;99(3):339–366. doi: 10.1085/jgp.99.3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halm D. R., Rechkemmer G. R., Schoumacher R. A., Frizzell R. A. Apical membrane chloride channels in a colonic cell line activated by secretory agonists. Am J Physiol. 1988 Apr;254(4 Pt 1):C505–C511. doi: 10.1152/ajpcell.1988.254.4.C505. [DOI] [PubMed] [Google Scholar]
  19. Hanrahan J. W., Tabcharani J. A. Inhibition of an outwardly rectifying anion channel by HEPES and related buffers. J Membr Biol. 1990 Jun;116(1):65–77. doi: 10.1007/BF01871673. [DOI] [PubMed] [Google Scholar]
  20. Hiratsuka T. Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives. Biochim Biophys Acta. 1982 Dec 17;719(3):509–517. doi: 10.1016/0304-4165(82)90240-9. [DOI] [PubMed] [Google Scholar]
  21. Hiratsuka T., Uchida K. Preparation and properties of 2'(or 3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, an analog of adenosine triphosphate. Biochim Biophys Acta. 1973 Oct 5;320(3):635–647. doi: 10.1016/0304-4165(73)90143-8. [DOI] [PubMed] [Google Scholar]
  22. Huettner J. E., Bean B. P. Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1307–1311. doi: 10.1073/pnas.85.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Karkaria C. E., Rosen B. P. Trinitrophenyl-ATP binding to the ArsA protein: the catalytic subunit of an anion pump. Arch Biochem Biophys. 1991 Jul;288(1):107–111. doi: 10.1016/0003-9861(91)90170-n. [DOI] [PubMed] [Google Scholar]
  24. Krishtal O. A., Marchenko S. M., Pidoplichko V. I. Receptor for ATP in the membrane of mammalian sensory neurones. Neurosci Lett. 1983 Jan 31;35(1):41–45. doi: 10.1016/0304-3940(83)90524-4. [DOI] [PubMed] [Google Scholar]
  25. Li M., McCann J. D., Liedtke C. M., Nairn A. C., Greengard P., Welsh M. J. Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature. 1988 Jan 28;331(6154):358–360. doi: 10.1038/331358a0. [DOI] [PubMed] [Google Scholar]
  26. Lukács G. L., Moczydlowski E. A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers. J Gen Physiol. 1990 Oct;96(4):707–733. doi: 10.1085/jgp.96.4.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Manning S. D., Williams A. J. Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers. J Membr Biol. 1989 Jul;109(2):113–122. doi: 10.1007/BF01870850. [DOI] [PubMed] [Google Scholar]
  28. Miller C. Competition for block of a Ca2(+)-activated K+ channel by charybdotoxin and tetraethylammonium. Neuron. 1988 Dec;1(10):1003–1006. doi: 10.1016/0896-6273(88)90157-2. [DOI] [PubMed] [Google Scholar]
  29. Moczydlowski E. G., Fortes P. A. Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP. J Biol Chem. 1981 Mar 10;256(5):2346–2356. [PubMed] [Google Scholar]
  30. Moczydlowski E. G., Fortes P. A. Inhibition of sodium and potassium adenosine triphosphatase by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenine nucleotides. Implications for the structure and mechanism of the Na:K pump. J Biol Chem. 1981 Mar 10;256(5):2357–2366. [PubMed] [Google Scholar]
  31. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983 Sep 8;305(5930):147–148. doi: 10.1038/305147a0. [DOI] [PubMed] [Google Scholar]
  32. Paulmichl M., Li Y., Wickman K., Ackerman M., Peralta E., Clapham D. New mammalian chloride channel identified by expression cloning. Nature. 1992 Mar 19;356(6366):238–241. doi: 10.1038/356238a0. [DOI] [PubMed] [Google Scholar]
  33. Rao R., Al-Shawi M. K., Senior A. E. Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate. J Biol Chem. 1988 Apr 25;263(12):5569–5573. [PubMed] [Google Scholar]
  34. Reinhardt R., Bridges R. J., Rummel W., Lindemann B. Properties of an anion-selective channel from rat colonic enterocyte plasma membranes reconstituted into planar phospholipid bilayers. J Membr Biol. 1987;95(1):47–54. doi: 10.1007/BF01869629. [DOI] [PubMed] [Google Scholar]
  35. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  36. Rosen B. P., Weigel U., Karkaria C., Gangola P. Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase. J Biol Chem. 1988 Mar 5;263(7):3067–3070. [PubMed] [Google Scholar]
  37. Schoumacher R. A., Shoemaker R. L., Halm D. R., Tallant E. A., Wallace R. W., Frizzell R. A. Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature. 1987 Dec 24;330(6150):752–754. doi: 10.1038/330752a0. [DOI] [PubMed] [Google Scholar]
  38. Singh A. K., Afink G. B., Venglarik C. J., Wang R. P., Bridges R. J. Colonic Cl channel blockade by three classes of compounds. Am J Physiol. 1991 Jul;261(1 Pt 1):C51–C63. doi: 10.1152/ajpcell.1991.261.1.C51. [DOI] [PubMed] [Google Scholar]
  39. Sneddon P., Westfall D. P., Fedan J. S. Cotransmitters in the motor nerves of the guinea pig vas deferens: electrophysiological evidence. Science. 1982 Nov 12;218(4573):693–695. doi: 10.1126/science.6291151. [DOI] [PubMed] [Google Scholar]
  40. Solc C. K., Wine J. J. Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells. Am J Physiol. 1991 Oct;261(4 Pt 1):C658–C674. doi: 10.1152/ajpcell.1991.261.4.C658. [DOI] [PubMed] [Google Scholar]
  41. Stutts M. J., Chinet T. C., Mason S. J., Fullton J. M., Clarke L. L., Boucher R. C. Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1621–1625. doi: 10.1073/pnas.89.5.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thomas P. J., Shenbagamurthi P., Ysern X., Pedersen P. L. Cystic fibrosis transmembrane conductance regulator: nucleotide binding to a synthetic peptide. Science. 1991 Feb 1;251(4993):555–557. doi: 10.1126/science.1703660. [DOI] [PubMed] [Google Scholar]
  43. Wang G. K. Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers. J Gen Physiol. 1988 Dec;92(6):747–765. doi: 10.1085/jgp.92.6.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Watanabe T., Inesi G. The use of 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate for studies of nucleotide interaction with sarcoplasmic reticulum vesicles. J Biol Chem. 1982 Oct 10;257(19):11510–11516. [PubMed] [Google Scholar]
  45. Welsh M. J. Electrolyte transport by airway epithelia. Physiol Rev. 1987 Oct;67(4):1143–1184. doi: 10.1152/physrev.1987.67.4.1143. [DOI] [PubMed] [Google Scholar]
  46. Winegar B. D., Lansman J. B. Voltage-dependent block by zinc of single calcium channels in mouse myotubes. J Physiol. 1990 Jun;425:563–578. doi: 10.1113/jphysiol.1990.sp018118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yellen G. Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J Gen Physiol. 1984 Aug;84(2):157–186. doi: 10.1085/jgp.84.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES