Mapping Conformational Changes of a Type IIb Na+/Pi Cotransporter by Voltage Clamp Fluorometry

Leila V Virkki,Heini Murer,Ian C Forster

doi:10.1074/jbc.m603861200

Abstract

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.

Highlights

IntroductionThe type II sodium-phosphate cotransporters (encoded by the solute carrier SLC34A gene family) are polytopic membrane proteins that mediate thermodynamically coupled transport of inorganic Pi and Naϩ across the cell membrane
The type II sodium-phosphate cotransporters are polytopic membrane proteins that mediate thermodynamically coupled transport of inorganic Pi and Naϩ across the cell membrane
We established the method of voltage clamp fluorometry (VCF)3 to gain new insights into putative conformational changes occurring during the transport cycle [4]

Summary

Introduction

The type II sodium-phosphate cotransporters (encoded by the solute carrier SLC34A gene family) are polytopic membrane proteins that mediate thermodynamically coupled transport of inorganic Pi and Naϩ across the cell membrane. Their physiological role is to facilitate cellular uptake of Pi by coupling it to the transmembrane electrochemical gradient. Trogenic and operate with a 3:1 Naϩ:HPO42Ϫ stoichiometry and translocate one net positive charge per transport cycle Using the new fluorescence data we modified our earlier kinetic model of the electrogenic NaPi-II transport cycle that we derived from the interpretation of steady state and presteady state substrate-dependent currents. In the S448C mutant the cotransport function is blocked after labeling, analysis of current and fluorescence data indicates that the protein is still able to bind substrate

Results

Discussion

Conclusion