SpringerProtocols: Abstract: Recording Currents from Channels and Transporters in Macropatches

Recording Currents from Channels and Transporters in Macropatches

By: Guiying Cui², Matthew D. Fuller³, Christopher H. Thompson², Zhi-Ren Zhang², Nael A. McCarty²

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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).

Affiliation(s): (2) School of Biology, Georgia Institute of Technology, Atlanta, GA
(3) Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA

Book Title: Patch-Clamp Analysis: Advanced Techniques

Series: Neuromethods | Volume: 38 | Pub. Date: Aug-02-2007 | Page Range: 353-371 | DOI: 10.1007/978-1-59745-492-6_12

Subject: Neuroscience

References

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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.

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.

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.

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.

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

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.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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