Abstract
The kinetics of a type IIb Na+-coupled inorganic phosphate (Pi) cotransporter (NaPi-IIb) cloned from mouse small intestine were studied using the two-electrode voltage clamp applied to Xenopus oocytes. In the steady state, mouse NaPi-IIb showed a curvilinear I-V relationship, with rate-limiting behavior only for depolarizing potentials. The Pi dose dependence was Michaelian, with an apparent affinity constant for Pi (\( {K_{\rm m}}^{\rm P_i} \)) of 10 ± 1 μM at −60 mV. Unlike for rat NaPi-IIa, \( {K_{\rm m}}^{\rm P_i} \) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na+ (\( {K_{\rm m}}^{\rm Na} \)) was 23 ± 1 mM at −60 mV, and the Na+ activation was cooperative with a Hill coefficient of approximately 2. Pre-steady-state currents were documented in the absence of Pi and showed a strong dependence on external Na+. The hyperpolarizing shift of the charge distribution midpoint potential was 65 mV/log[Na]. Approximately half the moveable charge was attributable to the empty carrier. A comparison of the voltage dependence of steady-state Pi-induced current and pre-steady-state charge movement indicated that for −120 mV ≤ V ≤ 0 mV the voltage dependence of the empty carrier was the main determinant of the curvilinear steady-state cotransport characteristic. External protons partially inhibited NaPi-IIb steady-state activity, independent of the titration of mono- and divalent Pi, and immobilized pre-steady-state charge movements associated with the first Na+ binding step.
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Notes
These proteins originate from three distinct solute carrier gene families SLC17 (type I, e.g., NPT1), SLC34 (type II, e.g., NaPi-II) and SLC20 (type III, e.g., Pit1/2) according to the Hugo gene classification for nomenclature (Hediger et al., 2004)
For rat NaPi-IIa and flounder NaPi-IIb isoforms, a fast component of charge movement has also been detected in the absence of Na+, leading to the proposal of an intermediate state for the empty carrier (Forster et al., 2000); however, for mouse NaPi-IIb, we were unable to detect such a component.
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Acknowledgement
This work was financially supported by grants awarded to J. B. and H. M. from the Swiss National Science Foundation and to H. M. from the Gebert Rüf Foundation ( http://www.grstiftung.ch). We thank Gerti Stange for expert technical assistance.
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Forster, I.C., Virkki, L., Bossi, E. et al. Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na+/Pi Cotransporter. J Membrane Biol 212, 177–190 (2006). https://doi.org/10.1007/s00232-006-0016-3
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DOI: https://doi.org/10.1007/s00232-006-0016-3