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.
The Journal of Membrane Biology – Springer Journals
Published: Nov 1, 1997
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