Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry - PubMed (original) (raw)
Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry
Sven Geibel et al. Proc Natl Acad Sci U S A. 2003.
Abstract
The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ( approximately 5%) on addition of extracellular K(+). When the enzyme was subjected to voltage jumps under Na(+)Na(+)-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E(1)P-E(2)P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na(+) by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K(+) concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K(+) concentrations. As a result, the stationary distribution between E(1) and E(2) states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na(+)Na(+) exchange as well as Na(+)K(+) turnover conditions.
Figures
Figure 1
(A) Albers–Post scheme for the Na+/K+-ATPase reaction cycle. The enzyme can assume two distinct conformations: E1 with ion binding sites facing the cytoplasm, and E2 with ion binding sites open to the extracellular space. The main electrogenic event was assigned to Na+ transport steps that are kinetically coupled to the E1P–E2P transition (underlaid in gray). (B) Na+/K+-ATPase α subunit modeled into the 1EUL structure of the SERCA Ca2+-ATPase (25) by using SWISSMODEL (courtesy of Jan B. Koenderink). Helix M5 and residue N790 (circle) are marked in red. Mutant N790C allowed for site-specific labeling by TMRM and yielded strong fluorescence changes in response to extracellular K+ or voltage pulses. Amino acids contributing to cation binding are colored as follows: E327, blue (helix M4); D776 and E779, red (helix M5); D804 and D808, green (helix M6). Two Ca2+ ions (yellow) from the 1EUL structure are also shown.
Figure 2
(A) Amino acids of the extracellular M5–M6 loop of the Na+/K+-ATPase. Amino acids of helices M5 and M6 are shown in black boxes. (B) Stationary pump currents of Na+/K+-ATPase single cysteine mutants at 0 mV in response to 10 mM K+. The dotted line indicates the stationary current level of the NaKWT and NaKØCys constructs. Data originated from three to five oocytes; values are means ± SE. (C) Fluorescence images of TMRM-labeled oocytes. (Upper) Uninjected control. (Lower) Oocyte injected with NaKØCys(N790C) α and β subunit cRNA. Labeling and illumination conditions were identical. (D) Parallel recording of pump current (Lower) and fluorescence change (Upper) from an oocyte expressing NaKØCys(N790C) in response to 10 mM K+ and 5 mM ouabain (see perfusion protocol) at 0 mV.
Figure 3
(A) Fluorescence change signals in the absence of K+ on voltage pulses from 0 mV to values as stated. (B) Voltage jump-induced transient currents, obtained as ouabain-sensitive difference currents (see Materials and Methods), recorded in parallel to traces in A. (C) Rate constants (reciprocal of time constants) for fluorescence changes (□) and transient currents (●) as depicted in A and B. Data are means ± SE from five oocytes. (D) Voltage dependence of fluorescence saturation values (□) and translocated charge (●) from experiments as shown in A and B. Data are means ± SE from five oocytes. Fits of a Boltzmann function are superimposed, yielding the following parameters: ΔF-V curve (dashed): V0.5 = −117 mV ± 1.4mV, zq = 0.76 ± 0.2; Q–V curve (solid): V0.5 = −110 mV ± 9 mV, zq = 0.85 ± 0.12.
Figure 4
(A_–_E) Voltage pulse-induced fluorescence responses from a continuous recording of an oocyte expressing NaKØCys(N790C) at different K+ concentrations. Fluorescence increase is indicated by the black arrow. (F) Control measurement after removal of extracellular K+. (G) Inhibition by 5 mM ouabain in the presence of 30 mM K+. (Inset) The applied voltage protocol.
Figure 5
(A) Voltage dependence of fluorescence saturation values at different K+ concentrations: 0 mM (□), 0.3 mM (○),1 mM (▵), 3 mM (▿),10 mM (⋄). (B) [K+] dependence of apparent rate constants of fluorescence signals on off voltage jumps to −80 mV. Data are means ± SE from three oocytes.
Similar articles
- Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.
Geibel S, Zimmermann D, Zifarelli G, Becker A, Koenderink JB, Hu YK, Kaplan JH, Friedrich T, Bamberg E. Geibel S, et al. Ann N Y Acad Sci. 2003 Apr;986:31-8. doi: 10.1111/j.1749-6632.2003.tb07136.x. Ann N Y Acad Sci. 2003. PMID: 12763772 - The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.
Dempski RE, Friedrich T, Bamberg E. Dempski RE, et al. J Gen Physiol. 2005 May;125(5):505-20. doi: 10.1085/jgp.200409186. J Gen Physiol. 2005. PMID: 15851504 Free PMC article. - Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes.
Dürr KL, Tavraz NN, Zimmermann D, Bamberg E, Friedrich T. Dürr KL, et al. Biochemistry. 2008 Apr 8;47(14):4288-97. doi: 10.1021/bi800092k. Epub 2008 Mar 15. Biochemistry. 2008. PMID: 18341291 - Voltage clamp fluorometry: combining fluorescence and electrophysiological methods to examine the structure-function of the Na(+)/K(+)-ATPase.
Dempski RE, Friedrich T, Bamberg E. Dempski RE, et al. Biochim Biophys Acta. 2009 Jun;1787(6):714-20. doi: 10.1016/j.bbabio.2009.03.021. Epub 2009 Apr 8. Biochim Biophys Acta. 2009. PMID: 19361481 Review. - Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase.
Jorgensen PL, Pedersen PA. Jorgensen PL, et al. Biochim Biophys Acta. 2001 May 1;1505(1):57-74. doi: 10.1016/s0005-2728(00)00277-2. Biochim Biophys Acta. 2001. PMID: 11248189 Review.
Cited by
- Displacement of the Na+/K+ pump's transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases.
Young VC, Artigas P. Young VC, et al. Proc Natl Acad Sci U S A. 2021 Feb 23;118(8):e2019317118. doi: 10.1073/pnas.2019317118. Proc Natl Acad Sci U S A. 2021. PMID: 33597302 Free PMC article. - Transient Electrical Currents Mediated by the Na+/K+-ATPase: A Tour from Basic Biophysics to Human Diseases.
Moreno C, Yano S, Bezanilla F, Latorre R, Holmgren M. Moreno C, et al. Biophys J. 2020 Jul 21;119(2):236-242. doi: 10.1016/j.bpj.2020.06.006. Epub 2020 Jun 12. Biophys J. 2020. PMID: 32579966 Free PMC article. Review. - The contribution of voltage clamp fluorometry to the understanding of channel and transporter mechanisms.
Cowgill J, Chanda B. Cowgill J, et al. J Gen Physiol. 2019 Oct 7;151(10):1163-1172. doi: 10.1085/jgp.201912372. Epub 2019 Aug 20. J Gen Physiol. 2019. PMID: 31431491 Free PMC article. Review. - Voltage Clamp Fluorometry of P-Type ATPases.
Dempski RE. Dempski RE. Methods Mol Biol. 2016;1377:281-91. doi: 10.1007/978-1-4939-3179-8_25. Methods Mol Biol. 2016. PMID: 26695040 Free PMC article. - Conformational changes represent the rate-limiting step in the transport cycle of maize sucrose transporter1.
Derrer C, Wittek A, Bamberg E, Carpaneto A, Dreyer I, Geiger D. Derrer C, et al. Plant Cell. 2013 Aug;25(8):3010-21. doi: 10.1105/tpc.113.113621. Epub 2013 Aug 20. Plant Cell. 2013. PMID: 23964025 Free PMC article.
References
- Post R L, Hegyvary C, Kume S. J Biol Chem. 1972;247:6530–6540. - PubMed
- Albers R W. Annu Rev Biochem. 1967;36:727–756. - PubMed
- Jorgensen P L. Biochim Biophys Acta. 1975;401:399–415. - PubMed
- Lutsenko S, Kaplan J H. J Biol Chem. 1994;269:4555–4564. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials