Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.     

Chemical rescue of a mutant protein-tyrosine kinase

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.     

Alterations in thin filament regulation induced by a human cardiac troponin T mutant that causes dilated cardiomyopathy are distinct from those induced by troponin T mutants that cause hypertrophic cardiomyopathy

We have compared the in vitro regulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the DeltaLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing DeltaLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase at pCa4.5. The Ca(2+) sensitivity of ATPase activation was increased (DeltapCa(50) = +0.2 pCa units) and cooperativity of Ca(2+) activation was virtually abolished. Equimolar mixtures of wild type and DeltaLys-210 troponin T gave a lower Ca(2+) sensitivity than with wild type, while maintaining the diminished ATPase activation at pCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca(2+) sensitivity was increased but there was no change in cooperativity. In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the DeltaLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli.     

A glutamine residue conserved in the neurotransmitter:sodium:symporters is essential for the interaction of chloride with the GABA transporter GAT-1

Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ～25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.     

Site-directed mutagenesis of rat cellular retinol-binding protein. Alteration in binding specificity resulting from mutation of glutamine 108 to arginine

Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein. A rat cDNA clone of CRBP was expressed in Escherichia coli. In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins. Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function. Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R). Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex. The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity. In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol. Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.     

Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q.

Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.     

Cytochrome b6 arginine 214 of Synechococcus sp. PCC 7002, a key residue for quinone-reductase site function and turnover of the cytochrome bf complex

Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.     

Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis

A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q, produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased specific activities of the mutants suggest that this regulatory site is close to the catalytic site.     

Role of the charge interaction between Arg(70) and Asp(120) in the Tn10-encoded metal-tetracycline/H(+) antiporter of Escherichia coli

We reported that the positive charge of Arg(70) is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Arg(70) may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp(66) and Asp(120), which are only two negatively charged residues located close to Arg(70) in putative secondary structure of TetA(B) and highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp(66) is essential, but Asp(120) is important for TetA(B) function. Surprisingly, when Asp(120) was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp(66) mutation. The charge-exchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg(2+)), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg(2+); however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg(2+), probably because the side chains at positions 70 and 120 are bridged with Hg(2+). The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10-phenanthroline). These results indicate that the positive charge of Arg(70) requires the negative charge of Asp(120) for neutralization, probably for properly positioning transmembrane segments in the membrane.     

Manganese selectivity of pmr1, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6. Residue Asp778 is essential for cation transport

We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca(2+)- and Mn(2+)-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment M6 of Pmr1. In phenotypic screens of Ca(2+) chelator and Mn(2+) toxicity reported separately (Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., and Rao, R. (2000) J. Biol. Chem. 275, XXXX-XXXX), D778A was a loss-of-function mutant apparently defective for transport of both Ca(2+) and Mn(2+), whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn(2+) transport. We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP. However, reverse phosphorylation from P(i) is preserved but is insensitive to inhibition by Ca(2+) or Mn(2+) ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca(2+) can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 micrometer, essentially identical to that of wild type (0.07 micrometer). Mn(2+) activation of ATP hydrolysis was half-maximal at 0.02 micrometer in wild type, establishing a normal selectivity profile of Mn(2+) > Ca(2+). Strikingly, Mn(2+)-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 micrometer. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln(783) in M6 may form a critical hydrophobic interaction with Val(335) in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.     

The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK(a) of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK(a) regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK(a) of the chromophore. R52 is found to flip out during the formation of PYP(M). The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK(a) regulation during the photocycle.     

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.     

Phenotype of a mechanosensitive channel mutant, mid-1, in a filamentous fungus, Neurospora crassa

In the yeast Saccharomyces cerevisiae, the MID1 (mating-induced death) gene encodes a stretch-activated channel which is required for successful mating; the mutant phenotype is rescued by elevated extracellular calcium. Homologs of the MID1 gene are found in fungi that are morphologically complex compared to yeast, both Basidiomycetes and Ascomycetes. We explored the phenotype of a mid-1 knockout mutant in the filamentous ascomycete Neurospora crassa. The mutant exhibits lower growth vigor than the wild type (which is not rescued by replete calcium) and mates successfully. Thus, the role of the MID-1 protein differs from that of the homologous gene product in yeast. Hyphal cytology, growth on diverse carbon sources, turgor regulation, and circadian rhythms of the mid-1 mutant are all similar to those of the wild type. However, basal turgor is lower than wild type, as is the activity of the plasma membrane H(+)-ATPase (measured by cyanide [CN(-)]-induced depolarization of the energy-dependent component of the membrane potential). In addition, the mutant is unable to grow at low extracellular Ca(2+) levels or when cytoplasmic Ca(2+) is elevated with the Ca(2+) ionophore A23187. We conclude that the MID-1 protein plays a role in regulation of ion transport via Ca(2+) homeostasis and signaling. In the absence of normal ion transport activity, the mutant exhibits poorer growth.     

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.     

Conserved asparagine residue located in binding pocket controls cation selectivity and substrate interactions in neuronal glutamate transporter

Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ～30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.     

Essential role of a single arginine of photosystem I in stabilizing the electron transfer complex with ferredoxin

PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K(d) of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K(d) value strongly depends on the charge of the residue substituting Arg-39: 0.22 microM for wild type, 1.5 microM for R39K, 56 microM for R39Q, and more than 100 microM for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K(d) value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin.     

Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T

In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal.     

The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.