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Recording Currents from Channels and Transporters in Macropatches

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Patch-Clamp Analysis

Part of the book series: Neuromethods ((NM,volume 38))

Abstract

This chapter describes methods for the study of ion channels and transporters by recording from membrane macropatches. While investigators have made use of many different cell types for such experiments, we focus here on studies of these proteins expressed exogenously in Xenopus oocytes. We rely on this model system in our laboratory for a number of reasons, including the fact that we are able to obtain seals of very high resistance, typically >150 GΩ. Where possible, we draw comparisons with the study of the same channels by other macroscopic recording techniques; where possible, we also compare results from macropatch experiments with results of similar experiments using single-channel recording. We provide examples of experiments with the following proteins: the human cystic fibrosis transmembrane conductance regulator (CFTR), the rabbit ClC-2 voltage-gated chloride channel, and a Na+/Ca2+ exchanger from Drosophila melanogaster (Calx1.2).

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References

  • Colquhoun, D. and Hawkes, A. G. (1995) The principles of the stochastic interpretation of ion-channel mechanisms, in Single-Channel Recording, 2nd ed. (Sakmann, B. and Neher, E., eds.), Plenum Press, New York, pp. 397–482.

    Google Scholar 

  • Cui, G., Song, B., and McCarty, N. A. (2004) Differential block of CFTR pore by three members of the sulphonylurea family. Biophys. J. 86, 586a.

    Google Scholar 

  • Fuller, M. D., Zhang, Z.-R., Cui, G., Kubanek, J., and McCarty, N. A. (2004) Inhibition of CFTR channels by a peptide toxin of scorpion venom. Am. J. Physiol. 287, C1328–C1341.

    Article  CAS  Google Scholar 

  • Fuller, M. D., Zhang, Z.-R., Cui, G., and McCarty, N. A. (2005) The block of CFTR by scorpion venom is state-dependent. Biophys. J. 89, 3960–3975.

    Article  PubMed  CAS  Google Scholar 

  • Gadsby, D. C., Vergani, P., and Csanády, L. (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440, 477–483.

    Article  PubMed  CAS  Google Scholar 

  • Hilgemann, D. W. (1995) The giant membrane patch, in Single-Channel Recording, 2nd ed. (Sakmann, B. and Neher, E., eds.), Plenum Press, New York, pp. 307–327.

    Google Scholar 

  • Hilgemann, D. W. (1996) The cardiac Na-Ca exchanger in giant membrane patches. Ann. N. Y. Acad. Sci. 779, 136–158.

    Article  PubMed  CAS  Google Scholar 

  • Hilgemann, D. W., Matsuoka, S., Nagel, G. A., and Collins, A. (1992) Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J. Gen. Physiol. 100, 905–932.

    Article  PubMed  CAS  Google Scholar 

  • Ikuma, M. and Welsh, M. J. (2000) Regulation of CFTR Cl channel gating by ATP binding and hydrolysis. Proc. Natl. Acad. Sci. USA 97, 8675–8680.

    Article  PubMed  CAS  Google Scholar 

  • Liman, E. R., Tytgat, J., and Hess, P. (1992) Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871.

    Article  PubMed  CAS  Google Scholar 

  • Linsdell, P. and Hanrahan, J. W. (1996) Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl channels expressed in a mammalian cell line, and its regulation by a critical pore residue. J. Physiol. (Cambr.) 496, 687–693.

    CAS  Google Scholar 

  • Linsdell, P. and Hanrahan, J. W. (1998) Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. J. Gen. Physiol. 111, 601–614.

    Article  PubMed  CAS  Google Scholar 

  • Machaka, K., Qu, Z., Kuruma, A., Hartzell, H. C., and McCarty, N. A. (2002) The endogenous Ca2+-activated Cl channel in Xenopus oocytes: a physiologically and biophysically rich model system, in Chloride Channels of Excitable and Non-excitable Cells (Fuller, C. M. and Benos, D. J., eds.), Academic Press, San Diego.

    Google Scholar 

  • McCarty, N. A. (2000) Permeation through the CFTR chloride channel. J. Exp. Biol. 203, 1947–1962.

    PubMed  CAS  Google Scholar 

  • McCarty, N. A., McDonough, S., Cohen, B. N., Riordan, J. R., Davidson, N., and Lester, H. A. (1993) Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl channel by two closely related arylamino-benzoates. J. Gen. Physiol. 102, 1–23.

    Article  PubMed  CAS  Google Scholar 

  • McCarty, N. A. and Zhang, Z.-R. (2001) Identification of a region of strong discrimination in the pore of CFTR. Am. J. Physiol. 281, L852–L867.

    CAS  Google Scholar 

  • McDonough, S., Davidson, N., Lester, H. A., and McCarty, N. A. (1994) Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron 13, 623–634.

    Article  PubMed  CAS  Google Scholar 

  • Omelchenko, A., Dyck, C., Hnatowich, M., et al. (1998) Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. J. Gen. Physiol. 111, 691–702.

    Article  PubMed  CAS  Google Scholar 

  • Quick, M. W., Naeve, J., Davidson, N., and Lester, H. A. (1992) Incubation with horse serum increases viability and decreases background neurotransmitter uptake in Xenopus oocytes. BioTechniques 13, 358–362.

    CAS  Google Scholar 

  • Riordan, J. R., Rommens, J. M., Kerem, B.-S., et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1072.

    Article  PubMed  CAS  Google Scholar 

  • Smith, S. S., Liu, X., Zhang, Z.-R. et al. (2001) CFTR: Covalent and noncovalent modification suggests a role for fixed charges in anion conduction. J. Gen. Physiol. 118, 407–431.

    Article  PubMed  CAS  Google Scholar 

  • Thompson, C. H., Fields, D. M., Olivetti, P. R., Fuller, M. D., Zhang, Z.-R., and McCarty, N. A. (2005) Inhibition of ClC-2 Cl channels by a peptide component of scorpion venom. J. Membr. Biol. 208, 65–76.

    Article  PubMed  CAS  Google Scholar 

  • Vergani, P., Lockless, S. W., Nairn, A. C., and Gadsby, D. C. (2005) CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433, 876–880.

    Article  PubMed  CAS  Google Scholar 

  • Vergani, P., Nairn, A. C., and Gadsby, D. C. (2003) On the mechanism of MgATP-dependent gating of CFTR Cl channels. J. Gen. Physiol. 120, 17–36.

    Google Scholar 

  • Welsh, M. J., Anderson, M. P., Rich, D. P., et al. (1992) Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation. Neuron 8, 821–829.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Z.-R., Cui, G., Liu, X., Song, B., Dawson, D. C., and McCarty, N. A. (2005a) Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel: one polypeptide forms one pore. J. Biol. Chem. 280, 458–468.

    PubMed  CAS  Google Scholar 

  • Zhang, Z.-R., Cui, G., Zeltwanger, S., and McCarty, N. A. (2004a) Time-dependent interactions of glibenclamide with CFTR: Kinetically complex block of macroscopic currents. J. Membr. Biol. 201, 139–155.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Z.-R., Song, B., and McCarty, N. A. (2005b) State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 280, 41997–42003.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Z.-R., Zeltwanger, S., and McCarty, N. A. (2000) Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes. J. Membr. Biol. 175, 35–52.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Z.-R., Zeltwanger, S., and McCarty, N. A. (2004b) Steady-state interaction of glibenclamide with CFTR: evidence for multiple sites. J. Membr. Biol. 199, 15–28.

    Article  PubMed  CAS  Google Scholar 

  • Zuñiga, L., Niemeyer, M. I., Varela, D., Catalán, M., Cid, L. P., and Sepúlveda, F. V. (2004) The voltage-dependent ClC-2 chloride channel has a dual gating mechanism. J. Physiol. 555, 671–682.

    Article  PubMed  Google Scholar 

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© 2007 Humana Press Inc., Totowa, NJ

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Cui, G., Fuller, M.D., Thompson, C.H., Zhang, ZR., McCarty, N.A. (2007). Recording Currents from Channels and Transporters in Macropatches. In: Walz, W. (eds) Patch-Clamp Analysis. Neuromethods, vol 38. Humana Press. https://doi.org/10.1007/978-1-59745-492-6_12

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  • DOI: https://doi.org/10.1007/978-1-59745-492-6_12

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-705-1

  • Online ISBN: 978-1-59745-492-6

  • eBook Packages: Springer Protocols

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