Patch Fluorometry

Abstract

Patch fluorometry is a biophysical technique that combines the power of patch clamping and optical recording, with the aim of correlating local conformational rearrangements in ion channel protein with the channel gating process. This is achieved through simultaneous recordings of fluorescence and current signals from the same population of ion channels in a membrane patch. The method can be applied to studies of ion channel structure–function relationship as well as membrane protein dynamics. Unlike nuclear magnetic resonance or other biophysical methods for structural study, patch fluorometry provides real‐time dynamic structural information from ion channels in their native membrane environment whereas they carry out their functions. Recent advances greatly expand the scope of this powerful method, allowing researchers to follow structural changes in channel protein with unprecedented spatial and temporal resolutions and accuracy.

Key Concepts:

  • Ion channels achieve their cell‐sensor functions through changes in conformation in response to physical or chemical stimuli.

  • Conformational changes in an ion channel protein do not always alter the current, and are thus invisible to patch clamp recording.

  • Fluorescence emission is highly sensitive to the local environment of fluorophores.

  • Site‐directed fluorophores serve as sensors for local structural changes.

  • Patch fluorometry allows direct correlation between channel protein conformational changes and the functional states of the channel.

Keywords: fluorescence microscopy; ion channel; patch-clamping; spectroscopy; protein dynamics; structure–function relationship; conformational rearrangements

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.

Genetically inserted fluorophores and fluorescent cAMP molecules. (a) Insertion sites of CFP or YFP on BK channel. Reproduced with permission from Miranda et al., © National Academy of Sciences. Red arrows indicate sites that tolerate an insertion. (b and c) Chemical structure of 8‐[DY‐547]‐AET‐cAMP and 8‐NBD‐cAMP, respectively.

Reproduced with permission from Kusch et al.2010, © Elsevier. The chromophore part is boxed.
Figure 4.

Structural model of the intracellular cyclic nucleotide‐binding domain of CNG channels, with the ligand and two fluorophore‐labelling sites highlighted.

close

References

Baruscotti M, Bottelli G, Milanesi R, DiFrancesco JC and DiFrancesco D (2010) HCN‐related channelopathies. Pflügers Archiv 460(2): 405–415.

Biskup C, Kusch J, Schulz E et al. (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446(7134): 440–443.

Blunck R, Starace DM, Correa AM and Bezanilla F (2004) Detecting rearrangements of shaker and NaChBac in real‐time with fluorescence spectroscopy in patch‐clamped mammalian cells. Biophysical Journal 86(6): 3966–3980.

Bykova EA, Zhang XD, Chen TY and Zheng J (2006) Large movement in the C terminus of CLC‐0 chloride channel during slow gating. Nature Structural and Molecular Biology 13(12): 1115–1119.

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, Snyder GE, Selvin PR and Bezanilla F (1999) Atomic scale movement of the voltage‐sensing region in a potassium channel measured via spectroscopy. Nature 402(6763): 809–813.

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.

Cheng W, Yang F, Takanishi CL and Zheng J (2007) Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. Journal of General Physiology 129(3): 191–207.

Cox DH, Cui J and Aldrich RW (1997) Allosteric gating of a large conductance Ca‐activated K+ channel. Journal of General Physiology 110(3): 257–281.

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.

Cui J and Aldrich RW (2000) Allosteric linkage between voltage and Ca(2+)‐dependent activation of BK‐type mslo1 K(+) channels. Biochemistry 39(50): 15612–15619.

DiFrancesco D and Tortora P (1991) Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature 351(6322): 145–147.

Giraldez T, Hughes TE and Sigworth FJ (2005) Generation of functional fluorescent BK channels by random insertion of GFP variants. Journal of General Physiology 126(5): 429–438.

Gonzalez C, Koch HP, Drum BM and Larsson HP (2010) Strong cooperativity between subunits in voltage‐gated proton channels. Nature Structural and Molecular Biology 17(1): 51–56.

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. Pflügers Archiv 391(2): 85–100.

Hille B (2001) Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates Inc.

Horrigan FT and Aldrich RW (2002) Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. Journal of General Physiology 120(3): 267–305.

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.

Jiang Y, Lee A, Chen J et al. (2002) Crystal structure and mechanism of a calcium‐gated potassium channel. Nature 417(6888): 515–522.

Kraemer A, Rehmann HR, Cool RH et al. (2001) Dynamic interaction of cAMP with the Rap guanine‐nucleotide exchange factor Epac1. Journal of Molecular Biology 306(5): 1167–1177.

Kusch J, Biskup C, Thon S et al. (2010) Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels. Neuron 67(1): 75–85.

Kusch J, Thon S, Schulz E et al. (2012) How subunits cooperate in cAMP‐induced activation of homotetrameric HCN2 channels. Nature Chemical Biology 8(2): 162–169.

Ludwig A, Zong X, Jeglitsch M, Hofmann F and Biel M (1998) A family of hyperpolarization‐activated mammalian cation channels. Nature 393(6685): 587–591.

Marni F, Wu S, Shah GM et al. (2012) Normal‐mode‐analysis‐guided investigation of crucial intersubunit contacts in the cAMP‐dependent gating in HCN channels. Biophysical Journal 103(1): 19–28.

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.

Milanesi R, Baruscotti M, Gnecchi-Ruscone T and DiFrancesco D (2006) Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. New England Journal of Medicine 354(2): 151–157.

Miranda P, Contreras JE, Plested AJ et al. (2013) State‐dependent FRET reports calcium‐ and voltage‐dependent gating‐ring motions in BK channels. Proceedings of the National Academy of Sciences of the USA 110(13): 5217–5222.

Nache V, Zimmer T, Wongsamitkul N et al. (2012) Differential regulation by cyclic nucleotides of the CNGA4 and CNGB1b subunits in olfactory cyclic nucleotide‐gated channels. Science Signaling 5(232): ra48.

Puljung MC and Zagotta WN (2008) 2164‐Pos site‐specific labeling of cysteine residues for patch‐clamp fluorometry. Biophysical Journal 94(1_MeetingAbstracts): 2164.

Puljung MC and Zagotta WN (2013) A secondary structural transition in the C‐helix promotes gating of cyclic nucleotide‐regulated ion channels. Journal of Biological Chemistry 288(18): 12944–12956.

Qiu F, Rebolledo S , Gonzalez C and Larsson HP (2013) Subunit interactions during cooperative opening of voltage‐gated proton channels. Neuron 77(2): 288–298.

Taraska JW and Zagotta WN (2007) Structural dynamics in the gating ring of cyclic nucleotide‐gated ion channels. Nature Structural and Molecular Biology 14(9): 854–860.

Taraska JW and Zagotta WN (2010) Fluorescence applications in molecular neurobiology. Neuron 66(2): 170–189.

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.

Wang L and Sigworth FJ (2009) Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature 461(7261): 292–295.

Wu S, Gao W, Xie C et al. (2012) Inner activation gate in S6 contributes to the state‐dependent binding of cAMP in full‐length HCN2 channel. Journal of General Physiology 140(1): 29–39.

Wu S, Vysotskaya ZV, Xu X et al. (2011) State‐dependent cAMP binding to functioning HCN channels studied by patch‐clamp fluorometry. Biophysical Journal 100(5): 1226–1232.

Wu Y, Yang Y, Ye S and Jiang Y (2010) Structure of the gating ring from the human large‐conductance Ca(2+)‐gated K(+) channel. Nature 466(7304): 393–397.

Xu X, Marni F, Wu S et al. (2012) Local and global interpretations of a disease‐causing mutation near the ligand entry path in hyperpolarization‐activated cAMP‐gated channel. Structure 20(12): 2116–2123.

Yang F, Cui Y, Wang K and Zheng J (2010) Thermosensitive TRP channel pore turret is part of the temperature activation pathway. Proceedings of the National Academy of Sciences of the USA 107(15): 7083–7088.

Yuan P, Leonetti MD, Pico AR, Hsiung Y and MacKinnon R (2010) Structure of the human BK channel Ca2+‐activation apparatus at 3.0 A resolution. Science 329(5988): 182–186.

Yuan P, Leonetti MD , Hsiung Y and MacKinnon R (2012) Open structure of the Ca2+ gating ring in the high‐conductance Ca2+‐activated K+ channel. Nature 481(7379): 94–97.

Zagotta WN, Olivier NB, Black KD et al. (2003) Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425(6954): 200–205.

Zheng J (2006) Patch fluorometry: shedding new light on ion channels. Physiology (Bethesda) 21: 6–12.

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.

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 (2004) Stoichiometry and assembly of olfactory cyclic nucleotide‐gated channels. Neuron 42(3): 411–421.

Further Reading

Lakowicz J (2006) Principles of Fluorescence Spectroscopy, 3rd edn. New York: Springer.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Zheng, Jie, and Yang, Fan(Sep 2013) Patch Fluorometry. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021386.pub2]