Electrophysiological characterization of the flounder type II Na+/Pi cotransporter (NaPi-5) expressed in Xenopus laevis oocytes.
The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/Pi cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state Pi-induced currents had a voltage-independent apparent K(m) for Pi 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 Pi 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 Pi (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 (V0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum Pi-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 Pi-independent, whereas changes in Na+ shifted V0.5 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.
Forster
I C
IC
Institute of Physiology, University of Zurich, Switzerland.
Wagner
C A
CA
Busch
A E
AE
Lang
F
F
Biber
J
J
Hernando
N
N
Murer
H
H
Werner
A
A
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D004553:Electric Conductivity; D004594:Electrophysiology; D005432:Flounder; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D009865:Oocytes / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D014982:Xenopus laevis / Q000378:metabolism
D002352:Carrier Proteins; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D012964:Sodium