Electrophysiological Characterization of the Flounder Type II Na+/P i Cotransporter (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, when superfusing with 96 mm Na⁺. The apparent K _m for Na⁺ at 1 mm P _i was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm 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 from 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 balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V _0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum P _i -suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec⁻¹. The voltage dependence of the relaxations was P _i -independent, whereas changes in Na⁺ shifted V _0.5 to −60 mV at 25 mm Na⁺. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na⁺. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na⁺ contribute to transmembrane charge movement. Received: 11 March 1997/Revised: 3 June 1997     

A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H+/Sucrose Cotransporter Expressed in Xenopus Oocytes

To elucidate the kinetic properties of the Arabidopsis H⁺/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range −180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H⁺ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (i ^H _max , i ^S _max for H⁺ and sucrose) and of the ligand concentrations eliciting half-maximal currents (K ^H _m , K ^S _m ). The i ^H _max and i ^S _max exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the K ^H _m and K ^S _m decreased with increasingly negative membrane potential. Furthermore, at any given voltage, i ^S _max increased and K ^S _m decreased as a function of the external H⁺ concentration. Eight six-state carrier models—which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier—were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton. Received: 23 January 1996/Revised: 16 April 1997     

Probing Transmembrane Topology of the High-Affinity Sodium/Glucose Cotransporter (SGLT1) with Histidine-Tagged Mutants

To reexamine the existing predictions about the general membrane topology of the high-affinity Na⁺/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 × Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na⁺/glucose cotransport activity at 0.1 mm d-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541–aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition. Received: 29 January 1999/Revised: 26 February 1999     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Membrane Topology Motifs in the SGLT Cotransporter Family

Homologues of the Na⁺/glucose cotransporter, the SGLT family, include sequences of mammalian, eubacterial, yeast, insect and nematode origin. The cotransported substrates are sugars, inositol, proline, pantothenate, iodide, urea and undetermined solutes. It is reasonable to expect that the SGLT family members share a similar or identical topology of membrane spanning elements, by virtue of their common ancestry and similar coupling of solute transport to downhill sodium flux. Here we examine their membrane topologies as deduced from diverse analyses of their primary sequences, and from their sequence correlations with the experimentally determined topology of the human Na⁺/glucose cotransporter SGLT1. Our analyses indicate that all family members share a common core of 13 transmembrane helices, but that some, like SGLT1 itself, have one additional span appended to the C-terminus, and still others, two. One bacterial member incorporates an additional span at the N-terminus. Sequence comparisons indicative of common ancestry of the SGLT and the [Na⁺+ Cl⁻] transporter families are introduced, and evaluated in light of their topologies. New evidence concerning the previously asserted common ancestry of SGLT1 and an N-acetylglucosamine permease of the bacterial phosphotransferase system is considered. Finally, we analyze observations which lead us to conjecture that the experimental strategy most commonly employed to reveal the topology of bacterial transporters (i.e., the fusion of reporter enzymes such as phoA alkaline phosphatase, beta-lactamase or beta-galactosidase, to progressively C-truncated fragments of the transporter) has often instead so perturbed local topology as to have entirely missed pairs of adjacent membrane spans. Received: 18 May 1996     

Regulation of Renal Na-HCO3 Cotransporter: VIII. Mechanism of Stimulatory Effect of Respiratory Acidosis

We examined the effect of respiratory acidosis on the Na-HCO₃ cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO₂ for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO₂ showed a significant increase in Na-HCO₃ cotransporter activity (expressed as % of control levels, 5 min: 142 ± 6, 2 hr: 144 ± 13, 4 hr: 145 ± 11, 24 hr: 150 ± 15, 48 hr: 162 ± 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO₂ for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO₂ for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO₂ for 5 min or 4 hr. In cells treated with vehicle, 10% CO₂ significantly increased cotransporter activity as compared to control cells exposed to 5% CO₂. This stimulation by 10% CO₂ was completely prevented by HAC or DHC at 5 min (5% CO₂: 1.8 ± 0.2, 10% CO₂: 2.6 ± 0.2, 10% CO₂+ HAC: 1.6 ± 0.2, 10% CO₂: +DHC: 2.0 ± 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO₂ for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO₃ cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis. Received: 28 March 1997/Revised: 22 December 1997     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Osmotic Equilibrium and Elastic Properties of Eel Intestinal Vesicles

Changes in volume of intestinal brush border membrane vesicles of the European eel Anguilla anguilla were measured as vesicles were exposed to media with different osmotic pressures. Preparing the vesicles in media of low osmotic pressure allowed the effects of a small hydrostatic pressure to become a significant factor in the osmotic equilibration. By applying LaPlace's law to relate pressure and volume and assuming a linear relation between membrane tension and area expansion, we estimate an initial membrane tension at 4.02 × 10⁻⁵ N cm⁻¹ and an area compressibility elastic modulus at 0.87 × 10⁻³ N cm⁻¹. The elastic modulus estimate falls in the low range of values reported for membranes from other tissues in other species. This lower modulus quantitatively accounts for why eel intestinal vesicles show measurable changes in volume in hypotonic media while rabbit kidney vesicles do not. Received: 28 January 1999/Revised: 15 June 1999     

Water Permeability of Brush Border Membrane Vesicles from Kidney Proximal Tubule

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability P _f evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. P _f values were of the order of 4 × 10⁻³ cm sec⁻¹ independent of the osmolarity of the preparation buffer. Arrhenius plots of P _f vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol⁻¹. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of P _f caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule. Received: 8 August 1996/Revised: 4 March 1997     

Detecting Changes in the Functional Constraints of Paralogous Genes

We describe a new procedure to determine whether regional alterations in the evolutionary constraints imposed on paralogous proteins have occurred. We used as models the A and B (alternatively called α and β) subunits of V/F/A-ATPases, originated by a gene duplication more than 3 billion years ago. Changes associated to three major splits (eubacteria versus Archaea-eukaryotes; Archaea versus eukaryotes; and among free-living bacteria and symbiotic mitochondria) were studied. Only in the first case, when we compared eubacterial or mitochondrial F-ATPases versus eukaryotic vacuolar V-ATPases or archaeal A-ATPases, constraint changes were observed. Modifications in the degree of regional constraining were not detected for the other two types of comparisons (V-ATPases versus A-ATPases and within F-ATPases, respectively). When the rates of evolution of the two subunits were compared, it was found that F-ATPases regulatory subunits evolved faster than catalytic subunits, but the opposite was true for A- and V-ATPases. Our results suggest that, even for universal and essential proteins, selective constraints may be occasionally altered. On the other hand, in some cases no changes were detected after periods of more than 2.2 billion years. Received: 24 February 2000 / Accepted: 12 August 2000     

Changes in Sodium or Glucose Filtration Rate Modulate Expression of Glucose Transporters in Renal Proximal Tubular Cells of Rat

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na⁺ are potential candidates. In this study we examined the role of glucose and Na⁺ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na⁺-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by ∼36%, SGLT1 by ∼20%, and GLUT2 by ∼100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by ∼100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by ∼150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na⁺ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule. Received: 14 July 2000/Revised: 8 March 2001     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998     

Adaptation by Corneal Epithelial Cells to Chronic Hypertonic Stress Depends on Upregulation of Na:K:2Cl Cotransporter Gene and Protein Expression and Ion Transport Activity

We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 μm bumetanide, proliferation measured as ³H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive ⁸⁶Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC. Received: 7 March 2000/Revised: 18 May 2000     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Presteady-State Currents of the Rabbit Na+/Glucose Cotransporter (SGLT1)

The rabbit Na⁺/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na⁺, membrane voltage V _m , phlorizin and temperature. Step changes in membrane voltage—from the holding V _h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K _i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V _m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q _max was 4–120 nC; V _0.5, the voltage for 50% Q _max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q _max and z were independent of V _h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V _0.5 towards more negative values. Decreasing [Na⁺] _o decreased Q _max, and shifted V _0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na⁺] _o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τ_max 8–20 msec. Decreasing [Na⁺] _o decreased τ_max, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τ_max. The maximum temperature coefficient Q ₁₀ for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na⁺/glucose cotransport indicate that charge-movements are due to Na⁺-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na⁺] _o is reduced; (iii) τ_max is decreased with decreasing [Na⁺] _o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na⁺] _o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996     

Cysteine Residues and the Structure of the Rat Renal Proximal Tubular Type II Sodium Phosphate Cotransporter (Rat NaPi IIa)

The rat renal Na/P _i cotransporter type IIa (rat NaP_i IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520. Received: 13 December 1999/Revised: 31 March 2000     

Formation of Folds and Vesicles by Dipalmitoylphosphatidylcholine Monolayers Spread in Excess

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents. Received: 18 May 2000/Revised: 20 November 2000