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 (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 相似文献   

Expression of rat renal sodium/phosphate cotransporter in Xenopus laevis oocytes.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.     

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

Endogenous L-glutamate transport in oocytes of Xenopus laevis.

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.     

Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts

alpha-Thrombin, a potent mitogen for the hamster fibroblast cell line CCL 39, stimulates by approximately 3-fold 86Rb+ uptake in a mutant lacking the Na+/H+ antiport activity (PS 120). The major component of this stimulated 86Rb+ (K+) uptake is a bumetanide-sensitive flux (IC50 = 0.4 microM), which accounts for 50% of total K+ uptake in Go-arrested cells and is approximately 4-fold stimulated by maximal thrombin concentrations (EC50 = 5 X 10(-4) units/ml). This bumetanide-sensitive 86Rb+ uptake represents a Na+/K+/Cl- cotransport, as indicated by its dependence on extracellular Na+ and Cl- and by the existence in PS 120 cells of a bumetanide-sensitive K+-dependent 22Na+ uptake. The stimulation reaches its maximum within 2 min, is reduced at acidic intracellular pH values (half-maximal at pHi = 6.8), and can also be induced, to a lesser extent, by EGF and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the effects of which are nearly additive. In contrast, preincubation with 12-O-tetradecanoylphorbol 13-acetate results in inhibition of thrombin- and EGF-induced stimulations, suggesting that activated protein kinase C might exert a feedback inhibitory control. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport is separated from the activation of the Na+/H+ antiport. However, activation of this cotransporter does not seem to play a major role in the mitogenic signaling pathway since its complete inhibition with bumetanide reduces only by 25-30% reinitiation of DNA synthesis.     

Functional expression of the Vibrio parahaemolyticus Na+/galactose (vSGLT) cotransporter in Xenopus laevis oocytes

We have successfully expressed a bacterial cotransporter in a functional form in the Xenopus laevis oocyte expression system. The goals were to compare the kinetics and selectivity of the cotransporter expressed in oocytes with those obtained in bacteria and in proteoliposomes, and to determine if it is possible to measure the electrical properties of the bacterial cotransporter expressed in oocytes. The Vibrio parahaemolyticus Na+/galactose cotransporter (vSGLT) expressed in oocytes has functional properties that are similar to those expressed in bacteria and those of the purified cotransporter reconstituted into liposomes. vSGLT is a Na+-dependent transporter that is saturable with Na+ (K(0.5)=17 mM) and D-galactose (K(0.5)=237 microM) and is sensitive to both D-fucose and phlorizin. In addition, vSGLT in oocytes shows a sugar specificity in the order of D-galactose >D-fucose > D-glucose, distinguishing it from the animal members of the Na+/glucose cotransporter family. The level of transport by vSGLT in oocytes is lower overall (V(max) approximately 10 pmol/oocyte/hour) compared to other plant and animal cotransporters (V(max) approximately 1000 pmol/oocyte/hour). The low level of expression does not permit us to carry out electrophysiological studies of the bacterial cotransporter. This study shows the potential and unique advantages of utilizing a eukaryotic oocyte expression system to study bacterial cotransporters.     

Sodium-inorganic phosphate cotransporter NaPi-IIb in the epididymis and its potential role in male fertility studied in a transgenic mouse model

Analysis by cDNA microarrays showed that in the murine epididymis, NaPi-IIb was the predominantly expressed epithelial isoform of the sodium-inorganic phosphate cotransporter and was markedly overexpressed in the proximal region in the infertile knockout (KO) compared to the fertile heterozygous (HET) c-ros transgenic mouse. The apparent up-regulation in the KO mouse confirmed by Northern and Western blot analyses could be explained by the absence of NaPi-IIb from the initial segment of the HET epididymis, as revealed by immunohistochemistry, and its presence on the epithelial brush border throughout the proximal epididymis of KO mice, where differentiation of the initial segment fails to occur. Both NaPi-IIb mRNA and protein were scarce or absent from the cauda epididymidis of both genotypes. A high content of inorganic phosphate was measured enzymatically in the HET cauda luminal fluid, with a 27% decrease in the KO mice. This decrease, presumably from a greater reabsorption of inorganic phosphate, particularly in the initial part of the KO epididymis, may disturb the normal process of sperm maturation in these infertile males. By contrast, no apparent consequences were observed for the transport of Na+ and Ca2+, the concentrations of which (approximately 26 mM and approximately 30 microM, respectively) were measured by microelectrodes to be identical in the caudal fluid from both genotypes.     

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent. MVs induced rapid mineralization approximately 3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required approximately 1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC(50)'s of 3-6 microM and approximately 10 microM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were approximately 1:30 for ACP, approximately 1:20 for HAP, approximately 1:3.3 for CPLX, and approximately 1:2.0 for MVs. MV mineralization was not found to be strictly Na(+)-dependent: substitution of Li(+) or K(+) for Na(+) had minimal effect; while N-methyl D-glucamine (NMG(+)) was totally inhibitory, choline(+) was clearly stimulatory. Na(+) substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG(+) totally blocked and choline(+) stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.     

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.     

The influence of inorganic phosphate on the activity of adenosine kinase

The enzyme adenosine kinase (AK; EC 2.7.1.20) shows a dependence upon inorganic phosphate (Pi) for activity. The degree of dependence varies among enzyme sources and the pH at which the activity is measured. At physiological pH, recombinant AK from Chinese hamster ovary (CHO) cells and AK from beef liver (BL) show higher affinities for the substrate adenosine (Ado), larger maximum velocities and lower sensitivities to substrate inhibition in the presence of Pi. At pH 6.2, both BL and CHO AK exhibit almost complete dependence on the presence of Pi for activity. The data show that both enzymes exhibit increasing relief from substrate inhibition upon increasing Pi and the inhibition of BL AK is almost completely alleviated by the addition of 50 mM Pi. The affinity of CHO AK for Ado increases asymptotically from K(m) 6.4 microM to a limit of 0.7 microM upon the addition of increasing Pi from 1 to 50 mM. The concentration of Ado necessary to invoke substrate inhibition also increases asymptotically from K(i) 32 microM to a limit of 69 microM at saturating concentrations of phosphate. In the presence of increasing amounts of Pi, the maximal velocity of activity increases hyperbolically. The effect that phosphate exerts on AK may be either to protect the enzyme from inactivation at high adenosine and H(+) concentrations or to stabilize substrate binding at the active site.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

Na(+)/glutamine (asparagine) cotransport by Staphylococcus lugdunensis and Corynebacterium amycolatum

Staphylococcus lugdunensis and Corynebacterium amycolatum each have a Na(+)/glutamine cotransporter that displays an ordered reaction sequence at the extracellular surface, with sodium binding (K(m) of 6.5 mM) before glutamine (K(m) of 50 microM). Asparagine is low-affinity substrate (K(m) approximately 1 mM) for each system.     

Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents

Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload.     

Voltage and substrate dependence of the inverse transport mode of the rabbit Na(+)/glucose cotransporter (SGLT1)

Properties of the cytoplasmic binding sites of the rabbit Na(+)/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using alpha-methyl D-glucopyranoside (alphaMDG) as a substrate. The apparent affinity for alphaMDG depends on the cytoplasmic Na(+) concentration and voltage. At 0 mV the K(M) for alphaMDG is 7 mM at 110 mM Na(+) and 31 mM at 10 mM Na(+). The apparent affinity for alphaMDG and Na(+) is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.     

Simultaneous measurements of ionic currents and leucine uptake at the amino acid cotransporter KAAT1 expressed in Xenopus laevis oocytes

The transport properties of the intestinal amino acid cotransporter KAAT1, heterologously expressed in Xenopus oocytes, were studied using simultaneous voltage-clamp and tritiated leucine uptake measurements. While addition of 1 mM leucine to oocytes kept at -80 mV in presence of Na(+) or K(+) caused an increase in holding current, in presence of Li(+) the current was reduced. Uptake measurements in voltage-clamp conditions showed that a comparable accumulation of amino acid occurred in all three ionic conditions and irrespective of the direction and amount of the current change. The ratio of moles of transferred charge to moles of transported amino acid ranges from 1.45 for K(+) to 3.52 for Li(+). A hypothetical interpretation involving the coexistence of two populations of transporters, one operating in the uncoupled mode and the other in the substrate transport mode is discussed.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

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 (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-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 P(i) 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 constants.