The Journal of General Physiology
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Published online Apr 24 2006. doi:10.1085/jgp.200609496
The Rockefeller University Press, 0022-1295 $8.00
JGP, Volume 127, Number 5, 539-555
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ARTICLE

 Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order



Leila V. Virkki, Heini Murer, and Ian C. Forster

Institute for Physiology and the Center for Integrative Human Physiology, University of Zurich, Zurich CH-8057, Switzerland

Correspondence to Ian C. Forster: Iforster{at}access.unizh.ch

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 ({Delta}F), which suggested that this site lies in an environment that is affected by conformational change in the protein. {Delta}F was substrate dependent (no {Delta}F 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.

Abbreviations used in this paper: MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methane thiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl)methane thiosulfonate; MTSET, (2-(trimethylammonium)ethyl methanethiosulfonate bromide); MTS-TMR: 2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate; NaPi-II, type II Na+/Pi cotransporter; VCF, voltage clamp fluorometry; WT, wild-type.


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