The sodium phosphate cotransporter family SLC34

This review summarizes the characteristics of the solute carrier family SLC34 that is represented by the type ll Na/Pi-cotransporters NaPi-lla (SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3). Other Na/Pi-cotransporters are described within the SLC17 and SLC20 families. Type ll Na/Pi-cotransporters are expressed in several tissues and play a major role in the homeostasis of inorganic phosphate. In kidney and small intestine, type ll Na/Pi-cotransporters are located at the apical sites of epithelial cells and represent the rate limiting steps for transepithelial movement of phosphate. Physiological and pathophysiological regulation of renal and small intestinal epithelial transport of phosphate occurs through alterations in the abundance of type ll Na/Pi-cotransporter

Topology of the Type IIa Na+/Pi Cotransporter

The type IIa Na+/Pi cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (Pi) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTO

Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes

Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na+-coupled phosphate cotransporter

A common structural feature characterises sodium-coupled inorganic phosphate cotransporters of the SLC34 family (NaPi-IIa/b/c): a pair of inverted regions in the N- and C-terminal halves of the protein. These regions are hypothesised to contain re-entrant domains that associate to allow alternating access of the substrates from either side of the membrane. To investigate if these domains interact during the NaPi-II transport cycle, we introduced novel cysteines at three functionally important sites associated with the predicted re-entrant domains of the flounder NaPi-IIb for the purpose of fluorescent labelling and cross-linking. Single and double mutants were expressed in Xenopus oocytes and their function analysed using electrophysiological and real-time fluorometric assays. The substitution at the cytosolic end of the first re-entrant domain induced a large hyperpolarizing shift in the voltage dependence of steady-state and presteady-state kinetics, whereas the two substitutions at the external face were less critical. By using Cu-phenanthroline to induce disulfide bridge formation, we observed a loss of transport activity that depended on the presence of sodium in the incubation medium. This suggested that external sodium increased the probability of NaPi-IIb occupying a conformation that favours interaction between sites in the re-entrant domains. Furthermore, voltage-dependent fluorescence data supported the hypothesis that a localised interaction between the two domains occurs that depends on the membrane potential and substrate present: we found that the fluorescence intensity reported by a labelled cysteine in one domain was dependent on the side chain substituted at a functionally critical site in the opposed domai

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

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±1μM 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 ste

Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. 32Pi uptake was strongly temperature-dependent and decreased by ∼80% at a temperature change from 25°C to 5°C. The corresponding activation energy (E a) was ∼14 kcalmol−1 for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM Pi and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the Pi- and PFA-induced changes in holding current decreased with temperature. E a at −100 mV for the cotransport and leak modes was ∼16 kcalmol−1 and ∼11 kcalmol−1, respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E a for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM Pi over a range of external Na+. The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na+-debinding rate compared with the other voltage-dependent rate constant

Transport Function of the Renal Type IIa Na+/Pi Cotransporter Is Codetermined by Residues in Two Opposing Linker Regions

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/Pi cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent Pi and Na+ affinities for Pi-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway