ArticleThe Journal of general physiology2006
Voltage clamp fluorometric measurements on a type II Na+-coupled Pi cotransporter: shedding light on substrate binding order.
Article in The Journal of general physiology, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 50 citations in OpenAlex.
- The "www" ofMembranes · 2022Review
- Mechanisms of phosphate transport.Nature reviews. Nephrology · 2019Review
- The molecular mechanism of SLC34 proteins: insights from two decades of transport assays and structure-function studies.Pflugers Archiv : European journal of physiology · 2019Review
- Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1.Proceedings of the National Academy of Sciences of the United States of America · 2017Article
- Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb.Biophysical journal · 2016Article
- A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.The Plant cell · 2016Article
- Correlating charge movements with local conformational changes of a Na(+)-coupled cotransporter.Biophysical journal · 2014Article
- Structural fold and binding sites of the human Na⁺-phosphate cotransporter NaPi-II.Biophysical journal · 2014Article
- The SLC34 family of sodium-dependent phosphate transporters.Pflugers Archiv : European journal of physiology · 2014Review
- Protonation controls ASIC1a activity via coordinated movements in multiple domains.The Journal of general physiology · 2014Article
- Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step.Pflugers Archiv : European journal of physiology · 2013Article
- Transposon-mediated alteration of TaMATE1B expression in wheat confers constitutive citrate efflux from root apices.Plant physiology · 2013Article
- Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na(+)-coupled phosphate cotransporter.Pflugers Archiv : European journal of physiology · 2011Article
- Effects of Na+ and H+ on steady-state and presteady-state currents of the human concentrative nucleoside transporter 3 (hCNT3).Pflugers Archiv : European journal of physiology · 2010Article
- HvALMT1 from barley is involved in the transport of organic anions.Journal of experimental botany · 2010Article
- Substrate interactions of the electroneutral Na+-coupled inorganic phosphate cotransporter (NaPi-IIc).The Journal of physiology · 2009Article
- Remarkable commonalities of electrogenic and electroneutral Na+-phosphate cotransporters.The Journal of physiology · 2009Article
- Elucidating conformational changes in the gamma-aminobutyric acid transporter-1.The Journal of biological chemistry · 2009Article
- Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/P i cotransporter.The Journal of membrane biology · 2007Article
- Conformational dynamics of hSGLT1 during Na+/glucose cotransport.The Journal of general physiology · 2006Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637-651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (DeltaF), which suggested that this site lies in an environment that is affected by conformational change in the protein. DeltaF was substrate dependent (no DeltaF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1-18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.
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