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

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

Steady-state kinetic characterization of the mouse B0AT1 sodium-dependent neutral amino acid transporter

The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B0AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L-Leu and Na+ generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient ~1) yielding a K0.5 for L-Leu of 1.16mM and for Na+ of 16mM at a holding potential of −50mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li+ can substitute partially for Na+ in the generation of L-Leu-evoked inward currents, whereas both Cl− and H+ concentrations influence its magnitude. The simultaneous measurement of charge translocation and L-Leu uptake in the same cell indicates that B0AT1 transports one Na+ per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na+, followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na+] along the renal proximal tubule both drives the uptake of neutral amino acids via B0AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cel

Steady-state kinetic characterization of the mouse B0AT1 sodium-dependent neutral amino acid transporter

The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B0AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L-Leu and Na+ generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient ~1) yielding a K0.5 for L-Leu of 1.16mM and for Na+ of 16mM at a holding potential of −50mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li+ can substitute partially for Na+ in the generation of L-Leu-evoked inward currents, whereas both Cl− and H+ concentrations influence its magnitude. The simultaneous measurement of charge translocation and L-Leu uptake in the same cell indicates that B0AT1 transports one Na+ per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na+, followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na+] along the renal proximal tubule both drives the uptake of neutral amino acids via B0AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cel