Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.     

Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K(+) channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K(+) and Ca(2+) conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.     

Amino acid transporter CAATCH1 is also an amino acid-gated cation channel.

CAATCH1 (cation-amino acid transporter/channel) is a recently cloned insect epithelial membrane protein related to mammalian Na(+)-, Cl(-)-coupled neurotransmitter transporters (Feldman, D. H., Harvey, W. R., and Stevens, B. R. (2000) J. Biol. Chem. 275, 24518-24526). In the present study we analyze the relationship between CAATCH1-mediated amino acid transport and ion fluxes by utilizing the Xenopus oocyte expression system in conjunction with electrophysiology and radiotracer uptake. Simultaneous flux measurements reveal that electrical currents and amino acid transport are thermodynamically uncoupled. This observation is supported by measuring significant uptake even in the absence of external alkali cations. Remarkably, CAATCH1-associated Na(+) or K(+) currents are large and do not saturate with voltage nor with cation concentration. These currents reverse in Nernstian fashion, thereby conferring channel activity in CAATCH1. Upon step-changes in the membrane potential, CAATCH1-expressing oocytes exhibit transient currents. Detailed analyses of these transients in the absence and presence of amino acids reveal direct ligand-protein interaction, demonstrating that binding by different amino acids (e.g. proline, threonine, methionine) differentially affects the state probability of CAATCH1 but has no effect on the maximal charge movement (Q(max)). Together these data suggest that CAATCH1 is a multifunction membrane protein that mediates thermodynamically uncoupled amino acid uptake but functions predominantly as an amino acid-gated alkali cation channel.     

Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism

Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16–23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.     

Amino acid-dependent increase in hepatic system N activity is linked to cell swelling

Treatment of cultured rat hepatocytes with certain amino acids stimulates the activity of the System N transporter. The present report investigates the mechanism by which the stimulatory amino acids elicit their effect. Activation of System N-mediated transport by amino acids is rapid, cycloheximide-insensitive, and involves neither trans-stimulation nor recruitment of additional carriers to the plasma membrane. In addition, the activation is Na(+)-dependent, supporting the related observation that the most effective stimulatory amino acids are substrates of Na(+)-dependent transport Systems A, ASC, and N whereas substrates of Na(+)-independent System L and non-amino acid metabolites are ineffective. The data suggest that active accumulation of amino acids via Na(+)-dependent carriers is necessary for the activation to occur. The amino acid-dependent stimulation is blocked in a concentration-dependent manner by increasing extracellular K+. Treatment of hepatocytes with an amino acid such as asparagine causes cell swelling and stimulation of System N activity; both of these effects are reduced by hypertonic media. Furthermore, swelling of rat hepatocytes with hypotonic media mimics the System N-stimulatory effects of asparagine. Among the Na(+)-dependent amino acid transport systems present in rat hepatocytes, System N is stimulated preferentially by amino acid-containing or hypotonic media. Collectively, these results demonstrate that cell swelling is a prerequisite for the amino acid-dependent activation of the hepatic System N transporter.     

Activity-dependent reversible inactivation of the general amino acid permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.     

Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.     

Are proton symports in yeast directly linked to H(+)-ATPase acidification?

Transport of amino acids in Saccharomyces cerevisiae is an H(+)-driven secondary active transport. Inhibitors of the plasma membrane H(+)-ATPase, particularly heavy water, diethylstilbestrol and suloctidil, were shown to affect the H(+)-extruding ATPase activity as well as the ATP-hydrolyzing activity, to a similar degree as they inhibited the transport of amino acids. The inhibitors had virtually no effect on the membrane electric potential or on the delta pH which constitute the thermodynamically relevant source of energy for these transports. Transport of acidic amino acids was affected much more than that of the neutral and especially of the basic ones. The effects were greater with higher amino acid concentrations. All this is taken as evidence that the amino acid carriers respond kinetically to the presence of protons directly at the membrane site where they are extruded by the H(+)-ATPase, rather than to the overall protonmotive force.     

Site-directed mutagenesis studies of rat steroid 5alpha-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5alpha-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K(m)(NADPH) and an approximately fourfold decrease in the V(max) with no change in K(m)(T). The mutation G185W resulted in a fivefold decrease in K(m)(NADPH) and an eightfold decrease in V(max), with no change in K(m)(T), whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V(max), an approximately 20-fold increase in K(m)(T) and no alteration in K(m)(NADPH), whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.     

Interactions between charged amino acid residues within transmembrane helices in the sulfate transporter SHST1

The aim of this study was to identify charged amino acid residues important for activity of the sulfate transporter SHST1. We mutated 10 charged amino acids in or near proposed transmembrane helices and expressed the resulting mutants in a sulfate transport-deficient yeast strain. Mutations affecting four residues resulted in a complete loss of sulfate transport; these residues were D107 and D122 in helix 1 and R354 and E366 in helix 8. All other mutants showed some reduction in transport activity. The E366Q mutant was unusual in that expression of the mutant protein was toxic to yeast cells. The R354Q mutant showed reduced trafficking to the plasma membrane, indicating that the protein was misfolded. However, transporter function (to a low level) and wild-type trafficking could be recovered by combining the R354Q mutation with either the E175Q or E270Q mutations. This suggested that R354 interacts with both E175 and E270. The triple mutant E175Q/E270Q/R354Q retained only marginal sulfate transport activity but was trafficked at wild-type levels, suggesting that a charge network between these three residues may be involved in the transport pathway, rather than in folding. D107 was also found to be essential for the ion transport pathway and may form a charge pair with R154, both of which are highly conserved. The information obtained on interactions between charged residues provides the first evidence for the possible spatial arrangement of transmembrane helices within any member of this transporter family. This information is used to develop a model for SHST1 tertiary structure.     

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.     

Highly conserved asparagine 82 controls the interaction of Na+ with the sodium-coupled neutral amino acid transporter SNAT2

The neutral amino acid transporter 2 (SNAT2), which belongs to the SLC38 family of solute transporters, couples the transport of amino acid to the cotransport of one Na(+) ion into the cell. Several polar amino acids are highly conserved within the SLC38 family. Here, we mutated three of these conserved amino acids, Asn(82) in the predicted transmembrane domain 1 (TMD1), Tyr(337) in TMD7, and Arg(374) in TMD8; and we studied the functional consequences of these modifications. The mutation of N82A virtually eliminated the alanine-induced transport current, as well as amino acid uptake by SNAT2. In contrast, the mutations Y337A and R374Q did not abolish amino acid transport. The K(m) of SNAT2 for its interaction with Na(+), K(Na(+)), was dramatically reduced by the N82A mutation, whereas the more conservative mutation N82S resulted in a K(Na(+)) that was in between SNAT2(N82A) and SNAT2(WT). These results were interpreted as a reduction of Na(+) affinity caused by the Asn(82) mutations, suggesting that these mutations interfere with the interaction of SNAT2 with the sodium ion. As a consequence of this dramatic reduction in Na(+) affinity, the apparent K(m) of SNAT2(N82A) for alanine was increased 27-fold compared with that of SNAT2(WT). Our results demonstrate a direct or indirect involvement of Asn(82) in Na(+) coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.     

Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.     

Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.     

Functional analysis of an inosine-guanosine transporter from Leishmania donovani. The role of conserved residues,aspartate 389 and arginine 393

Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Delta ldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp389 and Arg393 interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/R393D localized properly to the plasma membrane, while neither D389R/R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp389 is critical for optimal transporter function and that a positively charged or polar residue at Arg393 is essential for proper expression of LdNT2 at the plasma membrane.     

Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions

Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H(+) and Cl(-) transport as well as in nucleotide binding. The H(+) transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their H(+) transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H(+) transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H(+) transport 80% and only 20%, respectively. These mutants retain full Cl(-) transport activity. The results indicate that D210 participates in H(+) uptake at the cytosolic side and D27 in H(+) translocation through the membrane. Differently, E167Q has lost Cl(-) transport activity but retains the ability to transport H(+). The separate inactivation of H(+) and Cl(-) transport argues against the fatty acid anion transport mechanism of H(+) transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but not of NDP binding.     

The serine/threonine kinases SGK1, 3 and PKB stimulate the amino acid transporter ASCT2

The human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2/SLC1A5) plays an important role in the transport of neutral amino acids in epithelial cells. The serine and threonine kinases SGK1-3 and protein kinase B have been implicated in the regulation of several members of the SLC1 transporter family by enhancing their plasma membrane abundance. The present study explored whether those kinases modulate hASCT2. In Xenopus oocytes heterologously expressing hASCT2, coexpression of constitutively active (S422D)SGK1, (S419D)SGK3 or (T308DS473D)PKB upregulated the transporter activity. The stimulation requires the catalytical activity of the kinases since the inactive mutants (K127N)SGK1, (K191N)SGK3, and (T308AS473A)PKB failed to modulate the transporter. According to kinetic analysis and chemiluminescence assays, SGK1 and SGK3 modulate hASCT2 by enhancing the transporter abundance in the plasma membrane. As SGK1, 3 and PKB are activated by insulin and IGF1, the described mechanisms presumably participate in the hormonal stimulation of cellular amino acid uptake.