Abstract
Understanding the function of ion channels is a major goal of molecular neurophysiology. While standard electrophysiological methods are invaluable tools to investigate the gating of ion channels, the structural rearrangements that mediate the way a channel senses physiological signals and opens and closes its gates cannot be measured electrically in a direct way. Here, we describe a method, based on site-specific labeling of a channel of interest with an environmentally sensitive fluorophore, which makes it possible to monitor conformational changes of ion channels in biological membranes in real time.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mannuzzu LM, Moronne MM, Isacoff EY (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271:213–216
Zheng J, Zagotta WN (2000) Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron 28:369–374
Asamoah OK, Wuskell JP, Loew LM, Bezanilla F (2003) A fluorometric approach to local electric field measurements in a voltage-gated ion channel. Neuron 37:85–97
Cohen BE, Pralle A, Yao X, Swaminath G, Gandhi CS, Jan YN et al (2005) A fluorescent probe designed for studying protein conformational change. Proc Natl Acad Sci U S A 102:965–970
Glauner KS, Mannuzzu LM, Gandhi CS, Isacoff EY (1999) Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813–817
Cha A, Snyder GE, Selvin PR, Bezanilla F (1999) Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809–813
Loots E, Isacoff EY (2000) Molecular coupling of S4 to a K(+) channel’s slow inactivation gate. J Gen Physiol 116:623–636
Gandhi CS, Clark E, Loots E, Pralle A, Isacoff EY (2000) The orientation and molecular movement of a k(+) channel voltage-sensing domain. Neuron 40:515–525
Pathak MM, Yarov-Yarovoy V, Agarwal G, Roux B, Barth P, Kohout S et al (2007) Closing in on the resting state of the Shaker K(+) channel. Neuron 56:124–140
Sonnleitner A, Mannuzzu LM, Terakawa S, Isacoff EY (2002) Structural rearrangements in single ion channels detected optically in living cells. Proc Natl Acad Sci U S A 99:12759–12764
Blunck R, McGuire H, Hyde HC, Bezanilla F (2008) Fluorescence detection of the movement of single KcsA subunits reveals cooperativity. Proc Natl Acad Sci U S A 105:20263–20268
Lakowicz J (2006) Principles of fluorescence spectroscopy. Springer, New York
Loots E, Isacoff EY (1998) Protein rearrangements underlying slow inactivation of the Shaker K+ channel. J Gen Physiol 112:377–389
Gandhi CS, Loots E, Isacoff EY (2000) Reconstructing voltage sensor-pore interaction from a fluorescence scan of a voltage-gated K+ channel. Neuron 27:585–595
Cha A, Bezanilla F (1997) Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron 19:1127–1140
Mannuzzu LM, Isacoff EY (2000) Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence. J Gen Physiol 115:257–268
Pathak M, Kurtz L, Tombola F, Isacoff E (2005) The cooperative voltage sensor motion that gates a potassium channel. J Gen Physiol 125:57–69
Tombola F, Ulbrich MH, Kohout SC, Isacoff EY (2010) The opening of the two pores of the Hv1 voltage-gated proton channel is tuned by cooperativity. Nat Struct Mol Biol 17:44–50
Baker OS, Larsson HP, Mannuzzu LM, Isacoff EY (1998) Three transmembrane conformations and sequence-dependent displacement of the S4 domain in Shaker K+ channel gating. Neuron 20:1283–1294
Schönherr R, Mannuzzu LM, Isacoff EY, Heinemann SH (2002) Conformational switch between slow and fast gating modes: allosteric regulation of voltage sensor mobility in the EAG K+ channel. Neuron 35:935–949
Bruening-Wright A, Elinder F, Larsson HP (2007) Kinetic relationship between the voltage sensor and the activation gate in spHCN channels. J Gen Physiol 130:71–81
Chanda B, Bezanilla F (2002) Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. J Gen Physiol 120:629–645
Gonzalez C, Koch HP, Drum BM, Larsson HP (2010) Strong cooperativity between subunits in voltage-gated proton channels. Nat Struct Mol Biol 17:51–56
Kohout SC, Ulbrich MH, Bell SC, Isacoff EY (2008) Subunit organization and functional transitions in Ci-VSP. Nat Struct Mol Biol 15:106–108
Villalba-Galea CA, Sandtner W, Starace DM, Bezanilla F (2008) S4-based voltage sensors have three major conformations. Proc Natl Acad Sci U S A 105:17600–17607
Chang Y, Weiss DS (2002) Site-specific fluorescence reveals distinct structural changes with GABA receptor activation and antagonism. Nat Neurosci 5:1163–1168
Yao X, Parnot C, Deupi X, Ratnala VRP, Swaminath G, Farrens D et al (2006) Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor. Nat Chem Biol 2:417–422
Larsson HP, Tzingounis AV, Koch HP, Kavanaugh MP (2004) Fluorometric measurements of conformational changes in glutamate transporters. Proc Natl Acad Sci U S A 101:3951–3956
Dempski RE, Friedrich T, Bamberg E (2009) Voltage clamp fluorometry: combining fluorescence and electrophysiological methods to examine the structure–function of the Na(+)/K(+)-ATPase. Biochim Biophys Acta 1787:714–720
Blunck R, Starace DM, Correa AM, Bezanilla F (2004) Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells. Biophys J 86:3966–3980
Acknowledgement
This work was supported by NIH grant R01 NS35549 (E.Y.I.), and a postdoctoral fellowship of the Swiss National Science Foundation (SNSF, PA00P3_134163) (T.K.B.).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Berger, T.K., Isacoff, E.Y. (2015). Fluorescent Labeling for Patch-Clamp Fluorometry (PCF) Measurements of Real-Time Protein Motion in Ion Channels. In: Gautier, A., Hinner, M. (eds) Site-Specific Protein Labeling. Methods in Molecular Biology, vol 1266. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2272-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2272-7_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2271-0
Online ISBN: 978-1-4939-2272-7
eBook Packages: Springer Protocols