Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na+/Pi Cotransporter

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 (Km(pi)) of 10 +/- 1 microM: at -60 mV. Unlike for rat NaPi-IIa, (Km(pi)) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na(+) (Km(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 <or= V <or= 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.

Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

“…Numerical 23 simulations using this model predict the steady-state cotransport current ( Fig 4B) and presteady-state parameters (Fig 4C) that match closely with those obtained from experimental data, thereby validating its application. In addition to providing basic mechanistic insight into the differences in voltage-dependent kinetics for WT isoforms (e.g., (Forster, Virkki et al 2006) ), and a mechanistic basis for cation interactions (Forster, Biber et al 2000;Andrini, Meinild et al 2012), such a model can be used to identify critical partial reactions when the steady-state and presteady-state kinetics are altered by mutagenesis (see Section IV.A.4) Bacconi, Ravera et al 2007;Ghezzi, Murer et al 2009). At present, the model is severely underdetermined because the kinetics of substrate interaction on the cytosolic side are unknown.…”

“…(Busch, Waldegger et al 1994;Forster, Wagner et al 1997;Forster, Hernando et al 1998;Nalbant, Boehmer et al 1999;Segawa, Kaneko et al 2002;Graham, Nalbant et al 2003;Forster, Virkki et al 2006;Ghezzi, Murer et al 2009)). The apparent affinity constants for total P i (K 0.5 Pi ) are typically <100 µM (pH 7.4 and [Na + ] =100 mM), with the exception of (i) a zebra fish isoform (NaPi-IIb1) (K 0.5 Pi =250 µM) (Graham, Nalbant et al 2003), and (ii) the murine and rat NaPi-IIb that exhibit exceptionally high apparent P i affinities (K 0.5 Pi ≈10 µM) (Forster, Virkki et al 2006;Villa-Bellosta&Sorribas 2008). Furthermore, all SLC34 proteins preferentially transport divalent P i (HPO 4 2-) (Forster, Loo et al 1999;Bacconi, Virkki et al 2005).…”

“…Published estimates of φ(V) for SLC34 proteins lie in the range 4-13 s -1 (-60 mV, ∼20 o C) (Forster, Virkki et al 2006). This range encompasses that reported for other electrogenic cotransporters and is typically <100 s -1 (e.g.…”

“…Thus, we would expect higher values under mammalian physiological conditions (37 o C), given that the estimates of φ were obtained from oocyte data (18-20 o C). If we assume that the temperature dependence of P i -transport for the flounder NaPi-IIb (Bacconi, Ravera et al 2007) is representative for all isoforms (Q 10 =2.3), then the turnover estimated experimentally for rat NaPi-IIa (4.1 ± 0.6 s -1 , at -60 mV) (Forster, Virkki et al 2006) would be > 10 s -1 at 37 o C. The turnover rate for NaPi-IIc has not been experimentally determined.…”

The leak mode of type II Na+-Pi cotransporters

“…We have estimated the turnover rates for the cotransport cycle of electrogenic SLC34 proteins to lie in the range 4-13 s -1 (47). Therefore, if one net charge is translocated in the leak mode, the magnitude of I PFA suggests that the leak turnover rate will be <1 s -1 .…”