Electrophysiological Characterization of the Flounder Type II Na+/P
i
Cotransporter (NaPi-5) Expressed in Xenopus laevis Oocytes |
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Authors: | IC Forster CA Wagner AE Busch F Lang J Biber N Hernando H Murer A Werner |
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Institution: | (1) Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland, CH;(2) Institute of Physiology, Eberhard-Karls University, Tubingen, Germany, DE;(3) Max-Planck-Institute for Molecular Physiology, Dortmund, Germany, DE |
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Abstract: | The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties
of the type II Na+/P
i
cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P
i
-induced currents had a voltage-independent apparent K
m
for P
i
of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na+. The apparent K
m
for Na+ at 1 mm P
i
was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage
jumps elicited presteady-state relaxations in the presence of 96 mm Na+ which were suppressed at saturating P
i
(1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at
extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically
gave a midpoint voltage (V
0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly
with the maximum P
i
-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated
as 35 sec−1. The voltage dependence of the relaxations was P
i
-independent, whereas changes in Na+ shifted V
0.5 to −60 mV at 25 mm Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions
involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.
Received: 11 March 1997/Revised: 3 June 1997 |
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Keywords: | : Na+/Pi cotransporter — Proximal tubule — Voltage clamp — Steady-state — Presteady-state — Xenopus laevis oocyte expression |
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