Correlating Charge Movements with Local Conformational Changes of a Na-Coupled Cotransporter

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na⁺-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na⁺, providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

To investigate Na⁺ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na⁺-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na⁺ in the presence of various Mg²⁺ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na⁺ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na⁺ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na⁺ ions and followed by the conformational transition E₁/E₂ of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na⁺ ions are bound. Received: 8 October 1998/Revised: 29 December 1998     

Renal cortical basolateral Na+/HCO 3 − cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na⁺/HCO ₃ ^– cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na⁺ and HCO ₃ ^– , incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na⁺ and HCO ₃ ^– during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na⁺ and HCO ₃ ^– , PITC binds covalently to amino groups unprotected by substrates leaving the Na⁺/HCO ₃ ^– cotransporter available for specific labeling with FITC. Addition of NaHCO₃ to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4-diisothiocyanostilbene 2,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na⁺/HCO ₃ ^– cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Identification of Electric-Field-Dependent Steps in the Na,K-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.

Conformational changes in the human Na(+)/glucose cotransporter (hSGLT1) were examined using hSGLT1 Q457C expressed in Xenopus laevis oocytes and tagged with tetramethylrhodamine-6-maleimide (TMR6M). Na(+)/glucose cotransport is abolished in the TMR6M-labeled mutant, but the protein binds Na(+) and sugar [Loo et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 7789-7794]. Under voltage clamp the fluorescence of labeled Q457C was dependent on external cations. Increasing [Na(+)] increased fluorescence with a Hill coefficient of 2 and half-maximal concentration (K(Na)(0.5)) of 49 mM at -90 mV. Li(+) also increased fluorescence, whereas choline, tetraethylammonium, and N-methyl-D-glucamine did not. Fluorescence was increased by sugars with specificity: methyl alpha-D-glucopyranoside > D-glucose > D-galactose > D-mannitol. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in pre-steady-state charge movement and fluorescence. Charge vs voltage and fluorescence vs voltage curves followed Boltzmann relations with the same median voltage (V(0.5) = -50 mV), but the apparent valence was 1 for charge movement and 0.4 for fluorescence. V(0.5) for fluorescence and charge movement was shifted by -100 mV per 10-fold decrease in [Na(+)]. Under Na(+)-free conditions, there was a voltage-dependent change in fluorescence. Voltage-jump experiments showed that the maximal change in fluorescence increased 20% with sugar. These results indicate that Na(+), sugar, and membrane voltage change the local environment of the fluorophore at Q457C. Our interpretation of these results is (1) the conformational change of the empty transporter is voltage dependent, (2) two Na(+) ions can bind cooperatively to the protein before sugar, and (3) sugar binding induces a conformational change.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.     

Effects on Conformational States of the Rabbit Sodium/Glucose Cotransporter through Modulation of Polarity and Charge at Glutamine 457

The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na⁺ and glucose. Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C after reaction with methanethiosulfonate derivatives, but Na⁺ and sugar binding remain intact. To explore the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 were studied using chemical modification and the two-electrode voltage-clamp technique. Compared to wild-type SGLT1, Q457C exhibits ∼20-fold reduction in phloridzin affinity and preferential occupancy of an inward-facing state. Alkylation of Q457C by [(2-trimethylammonium) ethyl] methanethiosulphonate bromide, (MTSET), reverses these changes while blocking transport. Analysis of pre-steady-state currents in the absence of sugar yields three decay constants for each of Q457C, Q457C-MTSET and Q457R. Comparison of Q457C-MTSET and Q457R with Q457C and wild-type, reveals that inhibition of transport is accompanied by a decrease in magnitude and voltage-independence of the slow decay constant at negative potentials. But fast and medium decays remain unchanged. Computer simulation of transient currents suggests that introduction of positive charge at position 457 leads to a predominant outward rather than inward-facing conformational state. Taken together, the results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier.     

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     

Protonation controls ASIC1a activity via coordinated movements in multiple domains

Acid-sensing ion channels (ASICs) are neuronal Na⁺-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.     

Mechanism of the Na,K-ATPase Inhibition by MCS Derivatives

The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E₁ conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H⁺, Na⁺ or K⁺) in one of the two unspecific binding sites, and at high Na⁺ concentrations another Na⁺ ion was bound to the highly Na⁺-selective ion-binding site.     

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Functionally Important Residues in the Predicted 3<Superscript>rd</Superscript> Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na⁺/P_i, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na⁺ and one divalent P_i ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P_i transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P_i transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P_i-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na⁺-dependent P_i transport. Since electrogenic NaPi-II transports 3 Na⁺/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na⁺ binding sites in NaPi-IIa.     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

Identification of the First Sodium Binding Site of the Phosphate Cotransporter NaPi-IIa (SLC34A1)

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (P_i) in renal and intestinal epithelia. The transport cycle requires three Na⁺ ions and one divalent P_i to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na⁺ ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na⁺ ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li⁺ ions substitute for Na⁺ at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and P_i based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and ³²P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li⁺ ions to substitute for Na⁺ ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.     

Erratum to “Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States” [J. Mol. Biol. 386 (2009) 351-385]: Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H⁺ and Na⁺. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na⁺ and Li⁺ at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.     

Cation binding to brain plasma membranes: An evaluation of the use of anionic fluorescent probes

Cation binding to brain plasma membranes has been studied using anionic sulfonate fluorescent probes. Ion affinity sequences follow the order Mg²⁺ > Ca²⁺ ? K⁺ > Cs⁺ > Na⁺ > Li⁺. The order of effectiveness, in increasing probe fluorescence, is the reverse of the affinity sequence for ions of the same charge. The affinity orders for erythrocyte membranes and dipalmitoyl lecithin are Mg²⁺ > Ca²⁺ ? Cs⁺ > K⁺ > Na⁺ > Li⁺ and Mg²⁺ > Ca²⁺ ? Li⁺ > Na⁺ > K⁺ > Cs⁺. These sequence variations are related to the differences in the nature of the ion binding sites. Heterogeneity in ion binding sites is demonstrated. Evidence is presented for the role of proteins in binding hydrophobic probes. The problem of separating specific conformational effects on ion binding from nonspecific charge neutralization effects is discussed. Pyrene excimer fluoresence rules out the possibility of extensive changes in mobility in the lipid phase on cation binding. Tetrodotoxin has been shown to inhibit Li⁺-, Na⁺-, and K⁺-induced fluorescence enancements of 1-anilino-8-naphthalene sulfonate bound to brain membranes.     

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face

Summary In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na⁺ are electroneutral steps, and that release of Na⁺ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K⁺ indicate that binding and occlusion of K⁺ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na⁺ or K⁺ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (ion well). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP⁺) or tetraphenylborate (TPB^–), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP⁺ leads to a decrease of binding affinity for Na⁺ and K⁺, which is thought to result from the TPP^–-induced change of electric field strength in the access channel.Deceased (September 13, 1990).     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Determination of the Na(+)/glucose cotransporter (SGLT1) turnover rate using the ion-trap technique

The Na⁺/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of two Na⁺ ions and one glucose molecule using the so-called alternating access mechanism. According to this principle, each cotransporter molecule can adopt either of two main conformations: one with the binding sites accessible to the extracellular solution and one with the binding sites facing the intracellular solution. The turnover rate (TOR) is the number of complete cycles that each protein performs per second. Determination of the TOR has important consequences for investigation of the cotransport mechanism, as none of the rate constants involved in mediating transport in a given direction (conformational changes and binding and unbinding reactions) can be slower than the TOR measured under the same conditions. In addition, the TOR can be used to estimate the number of cotransporter molecules involved in generating a given ensemble activity. In this study, we obtain an independent estimation of the TOR for human SGLT1 expressed in Xenopus laevis oocytes applying the ion-trap technique. This approach detects the quantity of ions released in or taken up from the restricted space existing between the oocyte plasma membrane and the tip of a large ion-selective electrode. Taking advantage of the fact that hSGLT1 in the absence of Na⁺ can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s⁻¹ in the presence of 35 mM α-methyl-glucose at −150 mV (pH 5.5). For the same group of oocytes, a TOR of 13.3 ± 2.4 s⁻¹ was estimated under near-V_max conditions, i.e., in the presence of 90 mM Na⁺ and 5 mM α-methyl-glucose. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n = 14), and this activity was generated by an average of 3.6 ± 0.7 × 10¹¹ cotransporter molecules/oocyte.