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Biophys J
2004 Jun 01;866:3966-80. doi: 10.1529/biophysj.103.034512.
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Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells.
Blunck R
,
Starace DM
,
Correa AM
,
Bezanilla F
.
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Time-resolved fluorescence detection of site-directed probes is a major tool in the investigation of structure-function relationships of voltage-dependent ion channels. However, the technique has been limited so far to the Xenopus-oocyte system making it difficult to study proteins, like, e.g., the prokaryotic sodium channel NaChBac, whose expression in oocytes is insufficient or whose physiological functions are distorted in oocytes. To expand the application of site-directed fluorescence detection to these proteins, we used two techniques-semiconfocal epifluorescence and total internal reflection fluorescence-to detect time-resolved fluorescence changes from site-directed labeled proteins expressed in mammalian cells under patch-clamp conditions, and investigated the characteristics and limitations of the techniques. The voltage-sensitive dye, di-8-ANEPPS, was used to monitor control of the membrane voltage in epifluorescence and total internal reflection fluorescence. Fluorescence changes in patch-clamped cells were recorded from a Shaker channel mutant (M356C) labeled in the S3-S4 linker using semiconfocal epifluorescence. The gating kinetics and fluorescence changes were in accordance with previous studies using fluorescence spectroscopy in Xenopus-oocyte systems. We applied our technique to the prokaryotic sodium channel NaChBac. Voltage-dependent protein-rearrangements of S4 could be detected that are independent of inactivation. Comparison of the S3-S4 linker regions revealed structural differences to the KvAP voltage sensor. The results from the NaChBac channel point to structural requirements for the S3-S4 loop to generate a fluorescence signal.
Asamoah,
A fluorometric approach to local electric field measurements in a voltage-gated ion channel.
2003, Pubmed
Asamoah,
A fluorometric approach to local electric field measurements in a voltage-gated ion channel.
2003,
Pubmed
Blaustein,
Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel.
2000,
Pubmed
,
Xenbase
Burke,
Distinct structural requirements for clustering and immobilization of K+ channels by PSD-95.
1999,
Pubmed
Cha,
Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence.
1997,
Pubmed
,
Xenbase
Cha,
Structural implications of fluorescence quenching in the Shaker K+ channel.
1998,
Pubmed
Cha,
Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation.
1999,
Pubmed
,
Xenbase
Cha,
Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy.
1999,
Pubmed
Chahine,
Functional expression and properties of the human skeletal muscle sodium channel.
1994,
Pubmed
,
Xenbase
Chanda,
Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.
2002,
Pubmed
,
Xenbase
Gandhi,
The orientation and molecular movement of a k(+) channel voltage-sensing domain.
2003,
Pubmed
,
Xenbase
Gandhi,
Reconstructing voltage sensor-pore interaction from a fluorescence scan of a voltage-gated K+ channel.
2000,
Pubmed
Geibel,
Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.
2003,
Pubmed
,
Xenbase
Geibel,
Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.
2003,
Pubmed
,
Xenbase
Glauner,
Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.
1999,
Pubmed
,
Xenbase
Gonzalez,
Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker.
2000,
Pubmed
,
Xenbase
Gonzalez,
Periodic perturbations in Shaker K+ channel gating kinetics by deletions in the S3-S4 linker.
2001,
Pubmed
,
Xenbase
Jiang,
X-ray structure of a voltage-dependent K+ channel.
2003,
Pubmed
Jiang,
The principle of gating charge movement in a voltage-dependent K+ channel.
2003,
Pubmed
Jugloff,
Internalization of the Kv1.4 potassium channel is suppressed by clustering interactions with PSD-95.
2000,
Pubmed
Larsson,
Transmembrane movement of the shaker K+ channel S4.
1996,
Pubmed
,
Xenbase
Li,
Early fluorescence signals detect transitions at mammalian serotonin transporters.
2002,
Pubmed
,
Xenbase
Li,
An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.
2000,
Pubmed
,
Xenbase
Loew,
Design and characterization of electrochromic membrane probes.
1982,
Pubmed
Loo,
Conformational changes couple Na+ and glucose transport.
1998,
Pubmed
,
Xenbase
Mannuzzu,
Direct physical measure of conformational rearrangement underlying potassium channel gating.
1996,
Pubmed
,
Xenbase
Mannuzzu,
Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence.
2000,
Pubmed
,
Xenbase
Mathur,
Role of the S3-S4 linker in Shaker potassium channel activation.
1997,
Pubmed
,
Xenbase
Meinild,
Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.
2002,
Pubmed
,
Xenbase
Ren,
A prokaryotic voltage-gated sodium channel.
2001,
Pubmed
Riven,
Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy.
2003,
Pubmed
,
Xenbase
Schwarz,
Multiple potassium-channel components are produced by alternative splicing at the Shaker locus in Drosophila.
1988,
Pubmed
Schönherr,
Conformational switch between slow and fast gating modes: allosteric regulation of voltage sensor mobility in the EAG K+ channel.
2002,
Pubmed
,
Xenbase
Shoeb,
Cloning and functional characterization of the smooth muscle ether-a-go-go-related gene K+ channel. Potential role of a conserved amino acid substitution in the S4 region.
2003,
Pubmed
,
Xenbase
Smith,
Fast and slow voltage sensor movements in HERG potassium channels.
2002,
Pubmed
,
Xenbase
Sonnleitner,
Structural rearrangements in single ion channels detected optically in living cells.
2002,
Pubmed
,
Xenbase
Sund,
Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.
2000,
Pubmed
Sørensen,
Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4.
2000,
Pubmed
,
Xenbase
Tang,
Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel.
1997,
Pubmed
Vilardaga,
Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
2003,
Pubmed
Zheng,
Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry.
2000,
Pubmed
