Patch Fluorometry


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.



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Further Reading

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

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