ELECTROPHYSIOLOGICAL CHARACTERIZATION OF THE FLOUNDER TYPE-II NA+ P-ICOTRANSPORTER (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/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, w hen superfusing with 96 mM Na+. The apparent K-m for Na+ at 1 mM P-i w as strongly voltage dependent (increasing from 32 mM at -70 mV to 77 m M 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 fro m 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 ba lanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge t ransfer to a Boltzmann relationship typically gave a midpoint voltage (V-0.5) close to zero and an apparent valency of approximately 0.6. Th e maximum steady-state transport rate correlated linearly with the max imum P-i-suppressed charge movement, indicating that the relaxations w ere NaPi-5-specific. The apparent transporter turnover was estimated a s 35 sec(-1) The voltage dependence of the relaxations was P-i indepen dent, whereas changes in Na+ shifted V-0.5 to -60 mV at 25 mM Na+. Pro tons suppressed relaxations but contributed to no detectable charge mo vement in zero external Na+. The voltage dependent presteady-state beh avior of NaPi-5 could be described by a 3 state model in which the par tial reactions involving reorientation of the unloaded carrier and bin ding of Na+ contribute to transmembrane charge movement.