The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2).
The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range -140 </= V </= +40 mV. With Pi as the variable substrate, the apparent affinity constant (KmPi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. KmPi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, KmNa was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at -50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre-steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range -20 to -50 mV and an apparent valency of approximately 0.5 e-. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step.
Forster
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I
Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland. forster@physiol.unizh.ch
Hernando
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N
Biber
J
J
Murer
H
H
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D017245:Foscarnet / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008322:Mammals; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D010758:Phosphorus / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D014982:Xenopus laevis
D000998:Antiviral Agents; D002352:Carrier Proteins; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D027981:Symporters; D010758:Phosphorus; D017245:Foscarnet; D012964:Sodium