Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1

Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+) ions, one H(+) and the counter-transport of one K(+) ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph), whose three dimensional structure is known is also coupled to three Na(+) ions but only two Na(+) ion binding sites have been observed in the crystal structure of Glt(Ph). In order to fully utilize the Glt(Ph) structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph) and accurately determine the number and location of Na(+) ions coupled to transport. Several sites have been proposed for the binding of a third Na(+) ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph) and reveal a new site for the third Na(+) ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph), and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+) compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+) ion in Glt(Ph) and EAAT1.     

A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.     

Single-file diffusion and neurotransmitter transporters: Hodgkin and Keynes model revisited

Norepinephrine transporters (NETs) use the Na gradient to remove norepinephrine (NE) from the synaptic cleft of adrenergic neurons following NE release from the presynaptic terminal. By coupling NE to the inwardly directed Na gradient, it is possible to concentrate NE inside cells. This mechanism, which is referred to as co-transport or secondary transport (L?uger, 1991, Electrogenic Ion Pumps, Sinauer Associates) is apparently universal: Na coupled transport applies to serotonin transporters (SERTs), dopamine transporters (DATs), glutamate transporters, and many others, including transporters for osmolites, metabolites and substrates such as sugar. Recently we have shown that NETs and SERTs transport norepinephrine or serotonin as if Na and the transmitter permeated through an ion channel together 'Galli et al., 1998, PNAS 95, 13260-13265; Petersen and DeFelice, 1999, Nature Neurosci. 2, 605-610'. These data are paradoxical because it has been difficult to envisage how NE, for example, would couple to Na if these ions move passively through an open pore. An 'alternating access' model is usually evoked to explain coupling: in such models NE and Na bind to NET, which then undergoes a conformational change to release NE and Na on the inside. The empty transporter then turns outward to complete the cycle. Alternating-access models never afford access to an open channel. Rather, substrates and co-transported ions are occluded in the transporter and carried across the membrane. The coupling mechanism we propose is fundamentally different than the coupling mechanism evoked in the alternating access model. To explain coupling in co-transporters, we use a mechanism first evoked by 'Hodgkin and Keynes (1955) J. Physiol. 128, 61-88' to explain ion interactions in K-selective channels. In the Hodgkin and Keynes model, K ions move single-file through a long narrow pore. Their model accounted for the inward/outward flux ratio if they assumed that two K ions queue within the pore. We evoke a similar model for the co-transport of transmitter and Na. In our case, however, coupling occurs not only between like ions but also between unlike ions (i.e. the transmitter and Na ). We made a replica of the Hodgkin and Keynes mechanical model to test our ideas, and we extended the model with computer simulations using Monte Carlo methods. We also developed an analytic formula for Na coupled co-transport that is analogous to the single-file Ussing equation for channels. The model shows that stochastic diffusion through a long narrow pore can explain coupled transport. The length of the pore amplifies the Na gradient that drives co-transport.     

The SLC6/NSS family members KAAT1 and CAATCH1 have weak chloride dependence

KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta.1,2 They are members of the SLC6/NSS family, which groups membrane proteins that use Na+, K+, and Cl- gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT)3 has contributed significantly to understanding of the structure–function relationship in NSS proteins. Transport by AaLeuT is Cl- independent, whereas many neurotransmitter:sodium symporters like serotonin transporter (SERT), GABA transporter (GAT1), dopamine transporter, and norephinephrine transporter, among others, are strongly Cl- dependent.4 A single Cl- ion is found bound to one of the extracellular loops, EL2 in AaLeuT. The Cl- is 20 Ã… away from the Na and leucine binding sites, and thus it is unclear whether this Cl- binding site is physiologically important. The nature of the association of Cl- ions with these proteins during transport remains to be resolved. The Cl- binding site of two members of the family, the serotonin transporter SERT 4 and the GABA transporter GAT1 5, has been recently modelled on the basis of their functional properties and by structural homology to AaLeuT. The analyses have highlighted the role of a serine residue, that in the Cl--independent AaLeuT corresponds to Glu 290, and of an asparagine (Asn 286) that also contributes to the coordination of Na+ in the Na1 binding site of AaLeuT. KAAT1 and CAATCH1 are able to transport different amino acids depending on the contransported cation (Na+ or K+) but their Cl- dependence is not completely defined yet. With the aim to clarify the role exerted by chloride in SLC6/NSS transporters, the Cl--dependence of KAAT1 and CAATCH1 have been investigated by the expression in Xenopus laevis oocytes and the measurement of induced amino acid uptakes. Despite KAAT1 and CAATCH1 posses the same residue of serine (Ser342, KAAT1 numbering) present in strictly chloride dependent transporters, their transport activities resulted weakly Cl--dependent compared to GAT1. By analysis of the pH dependence of the KAAT1 and CAATCH1 transport activity, we obtained more information to define their (particular) peculiar Cl- dependence.     

Ion-binding properties of the ClC chloride selectivity filter

The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.     

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

Glutamate transport is coupled to the co-transport of 3 Na(+) and 1 H(+) followed by the counter-transport of 1 K(+). In addition, glutamate and Na(+) binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [(14)C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.     

Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants

The purpose of these experiments is to test whether the differences between normal and tetrodotoxin-resistant Na+ channels reside in the selectivity filter. To do this, we have compared the selectivity of batrachotoxin-activated channels for alkali cations, organic cations, and nonelectrolytes in two neuroblastoma clonal cell lines: N18, which has normal tetrodotoxin (TTX) sensitivity, and C9, which is relatively TTX-resistant. We have also studied the effect of H+ on Na+ permeability and on the interaction between TTX and its receptor site in both cell lines. There is no qualitative difference between the two cell lines in any of these properties. In both cell lines the batrachotoxin-activated Na+ channels have a selectivity sequence of Tl+ greater than Na+ greater than K+, guanidinium greater than Rb+ greater than Cs+, methylamine. Also, in both cell lines H+ blocks Na+ channels with a pKa of 5.5 and inhibits the action of TTX with the same pKa. These observations indicate that the selectivity filters of the Na+ channels in C9 and N18 do not differ significantly despite the 100-fold difference in TTX-affinity. Our selectivity studies of batrachotoxin-activated Na+ channels for both cell lines suggest that these toxin-activated Na+ channels have a limiting pore size of 3.8 x 6.0 A, as compared to a pore size of 3.0 x 5.0 A for potential-activated Na+ channels.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.     

Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.     

The dual-function glutamate transporters: structure and molecular characterisation of the substrate-binding sites

Glutamate transporters are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentrations below neurotoxic levels. These transporters also mediate a thermodynamically uncoupled chloride flux, activated by two of the molecules they transport, sodium and glutamate. Five eukaryotic glutamate transporters have been cloned and identified. They exhibit approximately 50% identity and this homology is even greater at the carboxyl terminal half, which is predicted to have an unusual topology. Determination of the topology shows that the carboxyl terminal part contains several transmembrane domains separated by two reentrant loops that are in close proximity to each other. We have identified several conserved amino acid residues in the carboxyl terminal half that play crucial roles in the interaction of the transporter with its substrates: sodium, potassium and glutamate. The conformation of the transporter gating the anion conductance is different from that during substrate translocation. However, there exists a dynamic equilibrium between these conformations.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Interaction of Tl+ with product complexes of fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH(4)(+)) will further enhance enzyme activity. Here, the interaction of Tl+ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl+ enhances catalysis by 20% with a K(a) of 1.3 mm and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg2+, Tl+, and reaction products, in which the concentration of Tl+ is 1 mm or less, reveal Mg2+ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl+. Intermediate concentrations of Tl+ (5-20 mm) displace Mg2+ from site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry with respect to bound orthophosphate. Loop 52-72 appears in a new conformational state, differing from its engaged conformation by disorder in residues 61-69. Tl+ does not bind to metal sites 1 or 2 in the presence of Mg2+, but does bind to four other sites with partial occupancy. Two of four Tl+ sites probably represent alternative binding sites for the site 3 catalytic Mg2+, whereas the other sites could play roles in monovalent cation activation.     

Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel

L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the amplitude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage clamp techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.     

Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

Ionic currents in the human serotonin transporter reveal inconsistencies in the alternating access hypothesis

We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.