Patch Fluorometry

Jie Zheng, The University of California, Davis, California, USA

Published online: December 2008

DOI: 10.1002/9780470015902.a0021386

Patch fluorometry is a technique aimed at correlating local conformational changes in ion channel protein with channel function. This is achieved through simultaneous recordings of fluorescence and current signals from ion channels in isolated membrane patches. The method can be applied to studies of ion channel structure–functional relationship and membrane protein dynamics.

Keywords: fluorescence microscopy; ion channel; patch clamping; imaging; protein dynamics

Figure 1. Schematic illustration of the configuration of voltage-clamp fluorometry and patch fluorometry.
Figure 2. Example methods for site-specific labelling of channel proteins with fluorophores. SH, sulfhydrol group of a cysteine; F, fluorophore; C, cysteine; X, any amino acid; MTS, methanethiosulfonate.
Figure 3. Structural model of the intracellular cyclic nucleotide-binding domain of CNG channels, with the ligand and two fluorophore-labelling sites highlighted.
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 References
    Biskup C, Kusch J, Schulz V et al. (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446(7134): 440–443.
    Cha A and Bezanilla F (1997) Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron 19(5): 1127–1140.
    Cha A and Bezanilla F (1998) Structural implications of fluorescence quenching in the Shaker K+ channel. Journal of General Physiology 112(4): 391–408.
    Cha A, Zerangue N, Kavanaugh M and Bezanilla F (1998) Fluorescence techniques for studying cloned channels and transporters expressed in Xenopus oocytes. Methods in Enzymology 296: 566–578.
    Craven KB, Olivier NB and Zagotta WN (2008) C-terminal movement during gating in cyclic nucleotide-modulated channels. Journal of Biological Chemistry 283(21): 14728–14738.
    Griffin BA, Adams SR and Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281(5374): 269–272.
    Hamill OP, Marty A, Neher E, Sakmann B and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391(2): 85–100.
    book Hille B (2001) Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates Inc.
    Islas LD and Zagotta WN (2006) Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence. Journal of General Physiology 128(3): 337–346.
    Matulef K, Flynn GE and Zagotta WN (1999) Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron 24(2): 443–452.
    Puljung MC and Zagotta WN (2008) 2164-Pos site-specific labeling of cysteine residues for patch-clamp fluorometry. Biophysical Journal 94(1_MeetingAbstracts): 2164.
    Taraska JW and Zagotta WN (2007) Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels. Nature Structural & Molecular Biology 14(9): 854–860.
    Trudeau MC and Zagotta WN (2004) Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry. Journal of General Physiology 124(3): 211–223.
    Varnum MD and Zagotta WN (1997) Interdomain interactions underlying activation of cyclic nucleotide-gated channels. Science 278(5335): 110–113.
    Zheng J (2006) Patch fluorometry: shedding new light on ion channels. Physiology (Bethesda) 21: 6–12.
    Zheng J, Varnum MD and Zagotta WN (2003) Disruption of an intersubunit interaction underlies Ca2+-calmodulin modulation of cyclic nucleotide-gated channels. Journal of Neuroscience 23(22): 8167–8175.
    Zheng J and Zagotta WN (2000) Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron 28(2): 369–374.
    Zheng J and Zagotta WN (2003) Patch-clamp fluorometry recording of conformational rearrangements of ion channels. Science's STKE 2003(176): PL7.
 Further Reading
    book Lakowicz J (2006) Principles of Fluorescence Spectroscopy, 3rd edn, p. 954. Springer: New York.
    Mannuzzu LM, Moronne MM and Isacoff EY (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271: 213–216.

Related Sub-Topics:

Ion Channels | Membrane Proteins
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Article Title: Patch Fluorometry
 
How to Cite close
Zheng, Jie(Dec 2008) Patch Fluorometry. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021386]