Abstract
Voltage clamp fluorometry has become a powerful tool to compare partial reactions of P-type ATPases such as the Na+,K+-ATPase and H+,K+-ATPase with conformational dynamics of these ion pumps. Here, we describe the methodology to heterologously express membrane proteins in X. laevis oocytes and site-specifically label these proteins with one or more fluorophores. Fluorescence changes are measured simultaneously with current measurements under two-electrode voltage clamp conditions.
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References
Mannuzu LM, Moronne MM, Isacoff EY (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271:213–216
Cha A, Bezanilla F (1997) Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron 19:1127–1140
Oelstrom K, Goldschen-Ohm MP, Holmgren M, Chanda B (2014) Evolutionarily conserved intracellular gate of voltage-dependent sodium channels. Nat Commun 5:3420
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
Koch HP, Larsson HP (2005) Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters. J Neurosci 25:1730–1736
Glauner KS, Mannuzu LM, Gandhi CS, Isacoff EY (1999) Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813–817
Geibel S, Kaplan JH, Bamberg E, Friedrich T (2003) Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry. Proc Natl Acad Sci U S A 100:964–969
Dempski RE, Lustig J, Friedrich T, Bamberg E (2008) Structural arrangement and conformational dynamics of the gamma subunit of the Na+/K+-ATPase. Biochemistry 47:257–266
Dempski RE, Hartung K, Friedrich T, Bamberg E (2006) Fluorometric measurements of intermolecular distances between the alpha and beta subunits of the Na+/K+-ATPase. J Biol Chem 281:36338–36346
Dempski RE, Friedrich T, Bamberg E (2005) The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme. J Gen Physiol 125:505–520
Geys S, Dempski RE, Bamberg E (2009) Ligand-dependent effects on the conformational equilibrium of the Na+, K+-ATPase as monitored by voltage clamp fluorometry. Biophys J 96:4561–4570
Dürr KL, Tavraz NN, Zimmermann D, Bamberg E, Friedrich T (2008) Characterization of Na, K-ATPase and H, K-ATPase enzymes with glycosylation-deficient b-subunit variants by voltage-clamp fluorometry in Xenopus oocytes. Biochemistry 47:4288–4297
Durr KL, Tavraz NN, Friedrich T (2012) Control of gastric H, K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes. PLoS One 7:e33645
Wagner CA, Friedrich B, Setiawan I, Lang F, Broer S (2000) The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 10:1–12
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
Biskup C, Kusch J, Schulz E, Nache V, Schwede F, Lehmann F, Hagen V, Benndorf K (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446:440–443
Lorenz C, Pusch M, Jentsch TJ (1996) Heteromultimeric CLC chloride channels with novel properties. Proc Natl Acad Sci U S A 92:13362–13366
Price EM, Lingrel JB (1988) Structure-function-relationships in the Na, K-ATPase alpha subunit-Site-directed mutagenesis of glutamine-111 and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27:8400–8408
Mourot A, Bamberg E, Rettinger J (2008) Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor. J Neurochem 105:413–424
Forster T (1948) Intermolecular energy migration and fluorescence. Ann Phys 2:55–75
Van Der Meer BW, Coker G, Chen SYD (1994) Resonance energy transfer: theory and data. Wiley, New York
Dale RE, Eisinger J, Blumberg WE (1979) The orientational freedom of molecular probes. Biophys J 26:161–194
Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer Academic/Plenum Publishers, New York
Cha A, Bezanilla F (1998) Structural implications of fluorescence quenching in the Shaker K+ channel. J Gen Physiol 112:391–408
Chen RF, Bowman RL (1965) Fluorescence polarization: measurement with ultraviolet-polarizing filters in spectrophotofluormeter. Science 147:729–732
Acknowledgements
The Dempski laboratory gratefully acknowledges the WPI Research Foundation and NIH (grant GM105964) for support. The author thanks Ryan Richards for critical reading of this manuscript.
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Dempski, R.E. (2016). Voltage Clamp Fluorometry of P-Type ATPases. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_25
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_25
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
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