Voltage clamp fluorometric measurements on a type II Na+-coupled Pi cotransporter: shedding light on substrate binding order.

The Journal of General Physiology
Leila VirkkiIan C Forster

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 u...Continue Reading

References

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Citations

Jan 9, 2007·The Journal of Membrane Biology·Tamara RadanovicHeini Murer
Apr 20, 2007·The Journal of Membrane Biology·Andrea BacconiIan C Forster
May 25, 2010·Pflügers Archiv : European journal of physiology·Edurne GorraitzMaria Pilar Lostao
Mar 9, 2011·Pflügers Archiv : European journal of physiology·Chiara GhezziIan C Forster
Mar 22, 2013·Pflügers Archiv : European journal of physiology·Monica PattiIan C Forster
Dec 20, 2013·Pflügers Archiv : European journal of physiology·Carsten A WagnerJürg Biber
Apr 14, 2009·The Journal of Biological Chemistry·Anne-Kristine MeinildNanna MacAulay
Nov 30, 2006·The Journal of General Physiology·Donald D F LooErnest M Wright
Dec 18, 2013·The Journal of General Physiology·Gaetano BonifacioStephan Kellenberger
Feb 24, 2010·Journal of Experimental Botany·Benjamin D GruberEmmanuel Delhaize
May 29, 2008·Clinical and Experimental Pharmacology & Physiology·Stephan A Pless, Joseph W Lynch
Feb 13, 2013·Annual Review of Physiology·Jürg BiberIan Forster
Mar 20, 2013·Molecular Aspects of Medicine·Ian C ForsterHeini Murer
Sep 7, 2006·Kidney International·I C ForsterH Murer
Mar 17, 2011·Advances in Chronic Kidney Disease·Ian ForsterAndreas Werner
Nov 17, 2007·The Plant Journal : for Cell and Molecular Biology·Peter KovermannEnrico Martinoia
Mar 28, 2012·The Journal of Medical Investigation : JMI·Shoji KuwaharaKen-ichi Miyamoto
Nov 1, 2017·Proceedings of the National Academy of Sciences of the United States of America·Edurne GorraitzDonald D F Loo
Mar 8, 2007·The Journal of Membrane Biology·Ian C ForsterJürg Biber
Aug 5, 2006·The Journal of Biological Chemistry·Leila V VirkkiIan C Forster
May 15, 2007·American Journal of Physiology. Cell Physiology·Silvia RaveraIan C Forster
Mar 31, 2012·American Journal of Physiology. Cell Physiology·Anne-Kristine Meinild, Ian C Forster
Jun 7, 2019·Nature Reviews. Nephrology·Moshe LeviHeini Murer
Jun 22, 2007·American Journal of Physiology. Renal Physiology·Leila V VirkkiIan C Forster
Nov 15, 2011·American Journal of Physiology. Cell Physiology·Olga AndriniIan C Forster
Oct 27, 2007·Biochemistry·Bruce A HirayamaErnest M Wright

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Methods Mentioned

BETA
Fluorescence

Software Mentioned

Axoscope
pClamp
Clampfit

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