A Novel Electrogenic Amino Acid Transporter Is Activated by K+ or Na+, Is Alkaline pH-dependent, and Is Cl−-independent*

A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acidtransporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product inXenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na+,Cl−-coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as l-proline, l-threonine, andl-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl−. Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K+ but preferring proline in the presence of Na+.

Here we report the cloning and characterization of a cDNA that encodes a new membrane protein, CAATCH1 (cation-anion-activated amino acid transporter/channel), from a caterpillar absorptive epithelium. When expressed in Xenopus oocytes, CAATCH1 acts as a Cl Ϫ -independent, and Na ϩ -or K ϩ -activated nutrient amino acid transporter that also yields currentvoltage relationships like those ascribed to ligand-modulated leakage behavior in DAT (8 -10). These unusual characteristics of CAATCH1, which are different from those of the structurally related KAAT1 (18), suggest that CAATCH1 plays a broader role in the Manduca midgut epithelium than that of a simple nutrient solute transporter. Moreover, CAATCH1 exhibits pH dependence and electrogenic activity that are appropriate for the caterpillar midgut in which the transapical voltage is about Ϫ240 mV in a highly alkaline luminal milieu (pH ϳ10.5) (19).

EXPERIMENTAL PROCEDURES
Cloning of CAATCH1 cDNA-Using a unique PCR 1 -based strategy, we obtained a cDNA clone from a Manduca sexta larval midgut library that had previously yielded several V-ATPase subunit clones (21). Our cloning strategy employed inosine degenerate primer touchdown PCR (22) using a mass-excised phagemid library as initial template, combined with specific primer-based PCR screening of phage lysates to isolate a clone that we designated CAATCH1. Subsequently, the functional CAATCH1 cRNA expression product was characterized in Xenopus oocytes.
The entire M. sexta midgut ZAP-XR cDNA library was mass-excised to generate plasmid dsDNA template, which was then used to generate unique target sequences that were isolated by degenerate primed PCR. An initial set of inosine-containing degenerate primers were designed to target conserved peptide motifs from invertebrate and vertebrate members of a subfamily of Na ϩ ,Cl Ϫ -dependent transporters serving various neurotransmitters and amino acids (23). The sense primer, S34 (5Ј-GGIAA(C/T)GTITGG(A/C)G(A/G/C/T)TT(C/T)CC-3Ј), was based on a GNVWRFP peptide motif, whereas the antisense primer, S21 (5Ј-IGC(A/G/T)ATIGCITC(A/G/C/T)GG(A/G)TA-3Ј), was based on a YP(D/ E)AIA peptide motif. Another sense primer, S22 (5Ј-GGIAA(C/T)GTIT-GG(G/T)G(A/G/C/T)TT(C/T)CC-3Ј), a tolerated alternative to S34, was also used in conjunction with S21 antisense primer for initial screening. "Touchdown" hot start PCR conditions (22) with Taq DNA polymerase (Roche Molecular Biochemicals) and 1.5 mM MgCl 2 were employed. Twenty cycles with decrementing annealing temperatures (60 -46°C, 0.7°C steps) were followed by 30 cycles at the final annealing temperature. For each cycle, denaturation was at 92°C for 40 s, annealing (60 -46°C during first 20 cycles, 46°C for final 30 cycles) was for 40 s, extension was at 60°C for 40 s, and then a final 75°C extension for 5 min was performed. The resulting 943-bp PCR fragment from the dsDNA library was TA-cloned into a pCR2.1 vector (Invitrogen) and sequenced; the fragment was related to various transporters (23), including the KAAT1 neutral amino acid transporter (18).
Based on the unique sequence of the 943-bp fragment, a second nested PCR primer set was designed for use in subsequent screening steps. This primer set was designed specifically to exclude KAAT1, while amplifying a unique 328-bp segment. In this case, sense (S25) was 5Ј-AACACTTGCTGCATCAGTCAC-3Ј and antisense (S26) was 5Ј-CT-CAAGGAGTTTCGCCCATTG-3Ј. The PCR cycling conditions were as follows: 40 cycles of denaturation at 92°C, 40 s; annealing at 57°C, 40 s; extension at 74°C, 30 s. A final 5-min extension step at 75°C was employed. The exclusion specificity of the S25/S26 set was confirmed by the failure to create any PCR product from negative control templates (e.g. purified full-length KAAT1 in plasmid DNA. However, we were able to generate the appropriate fragment from the cloned 943-bp template positive control. The S25/S26 set was used for subsequent phage lysate PCR screening steps and was used with the cloned 943-bp fragment to create a 328-bp digoxigenin-labeled (Roche Molecular Biochemicals) double-stranded DNA plaque hybridization probe for the isolation of a single clone.
Phage lysate screening (24) was initiated by infecting XL1-Blue MRFЈ host cells with the ZAP-XR library (5 ϫ 10 4 plaque-forming units total). Following plaque formation, SM buffer lysates were collected from the plate and screened by PCR to identify a positive 328-bp band using the S25/S26 primers. The positive lysates were further screened by PCR using vector T3 sense primer with S26 antisense primer, and four clones with long 5Ј-UTR sequences were obtained. The lysates positive for both S25/S26 and T3/S26 primer combinations were then replated at greater dilution, and the resulting lysates were rescreened by PCR. After three rounds of screening progressively smaller pools of clones, a positive lysate derived from plates seeded with 80 plaque-forming units was subsequently plated (ϳ200 plaque-forming units total) on a single 10-cm dish with XL1-Blue MRFЈ host cells, and a standard plaque lift was performed; the membrane (Hybond; Amersham Pharmacia Biotech) was probed using the 328-bp digoxigeninlabeled probe (above) hybridized under high stringency conditions (66°C), yielding five positive plaques. Plasmid DNA from two plaques was excised in vivo and cloned. One clone containing a ϳ3-kilobase insert (CAATCH1) was used in all subsequent analyses including sequencing and physiological studies. The complete coding sequence of CAATCH1, as well as 5Ј-and 3Ј-UTRs, was determined by primer walking the insert between M13 sites of the pBluescript vector (Stratagene).
DNA samples were sequenced in the DNA Sequencing Core Laboratory of the Florida Interdisciplinary Center for Biotechnology Research. Sequencing was carried out in both directions of the plasmid using Taq DyeDeoxy terminator and DyePrimer Cycle Sequencing protocols developed by Applied BioSystems using fluorescence-labeled dideoxynucleotides and primers. The labeled extension products were analyzed with an Applied BioSystems model 373A DNA sequencer.
Transcription and Expression of cRNA in Oocytes-The plasmid containing full-length CAATCH1 cDNA was linearized by XhoI digestion, and m7G(5Ј)ppp(5Ј)G-capped cRNA was synthesized in vitro by using the T3 RNA polymerase promoter with the mMessage mMachine (Ambion) kit. Xenopus laevis oocytes at stage V or VI were injected with 50 nl of water with or without 50 ng of cRNA and then incubated at 17°C in Barth's saline for 3-10 days.
Electrophysiology-Injected oocytes were superperfused (22°C) with modified ND96 medium (98 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM TAPS plus N-methyl-D-glucamine (NMG), pH 8.0) at a constant rate (ϳ3 ml/min unless otherwise noted) using a peristaltic pump. The pH of the medium was 8.0 unless otherwise noted. To determine the cationic specificity of CAATCH1, Na ϩ was completely replaced by K ϩ , N-methyl-D-glucamine ϩ , choline ϩ , Rb ϩ , or Li ϩ in all buffer salts. To determine its anionic specificity, gluconate Ϫ was completely replaced by Cl Ϫ in all buffer salts. Transmembrane currents were measured in intact oocytes using a two-electrode voltage clamp (Warner model OC725-B, Hamden, CT) with agar-bridged bath electrodes (25). Data were filtered at 8 Hz (Ϫ3 db Bessel filter), digitized at 20 Hz, and analyzed with our custom programs utilizing LabView software (National Instruments, Austin, TX). Current-voltage relations were generated using voltage steps or ramps (36 mV/s, 1.8 mV/point) between Ϫ150 and ϩ30 mV from a given holding potential (usually Ϫ60 mV unless noted). Substrate-dependent, current-voltage data were obtained by subtracting control current values measured in the absence of substrate. Leakage I-V data that represented CAATCH1-associated leakage currents exposed by a substrate, were computed by digital subtraction of current in the presence of leakage-exposing substrate from control current (I CON minus I S ). Transport-associated I-V data represent CAATCH1 currents appearing only in the presence of a solute substrate, computed by the inverse operation (I S minus I CON ). Experiments were replicated at least three times, with confirmation in at least two separate injection batches of oocytes. Data means were derived from n Ն 3 replicate measurements, and figures show representative plots.
Perioocyte pH Measurements-Real time measurements of perioocyte extracellular pH were obtained using a model MI-419-E/MV-ADPT pH microelectrode in a 21-gauge needle (Microelectrode, Inc., Bedford, NH) placed 50 m from the oocyte surface and referenced to the same agar-bridged reference electrode that was used for the voltage clamp circuit. Data captured with this pH microelectrode and LabView software gave a resolution of Ϯ0.003 pH units. For these experiments, the extracellular bathing solution consisted of 98 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , and 1 mM CAPSO/NaOH at pH 8.410. Media containing L-proline were also adjusted to pH 8.410 Ϯ 0.003. To deliberately maximize pH changes, we employed CAPSO buffer (pK 9.6) at a low buffer concentration and reduced the bath perfusion rate to 0.5 ml min Ϫ1 .
Materials-Buffer reagents were obtained from Sigma. Oligonucleotide primers were synthesized by the University of Florida DNA synthesis core facility.

RESULTS
A single clone was obtained with a full-length sequence of 2858 base pairs (CAATCH1; GenBank TM accession no. AF013963). It contains an open reading frame of 1899 base pairs that encodes a polypeptide with 633 amino acid residues ( Fig. 1) and has an unglycosylated relative molecular mass of 69,934 with pI ϭ 7.37. A 5Ј-UTR of 100 bases and a polyadenylated 3Ј-UTR of 859 bases flank the open reading frame. The predicted topology of CAATCH1 (27, 28) includes 12 putative transmembrane domains, with cytosolic N-and C-terminal segments rich in proline, as well as acidic and basic amino residues (Fig. 1A). Seven consensus phosphorylation sites are located within cytoplasmic loops, and two N-linked glycosylation sites exist on the extracellular loop between the third and fourth membrane-spanning segments. Scanning the Gen-Bank TM data base reveals that the sequence of CAATCH1 is related to Na ϩ ,Cl Ϫ -dependent solute transporters (e.g. human dopamine transporter hDAT; Fig. 1B) in species ranging from Caenorhabditis elegans to Homo sapiens with 35-45% nucleotide sequence identity and 35-39% amino acid sequence identity (ϳ50% similar); such transporters mediate the reuptake of dopamine, serotonin, norepinephrine, GABA, glycine, and proline (8 -10, 12, 23, 29 -32). The most closely related clone is the M. sexta KAAT1 neutral amino acid transporter (18) (Fig. 1) with overall 92% nucleotide identity and 90% amino acid identity (94% similarity) with CAATCH1. Amino acid differences are scattered throughout the sequence; however, conspicuous sequence differences are found within or near predicted transmembrane domains 6, 11, and 12 as well as in their adjacent hydrophilic, cytosolic C-and N-terminal regions. For example, between CAATCH1 residues Leu 496 and Ile 577 , 25 amino acids (or 31%) diverge, often with nonconservative differences. Moreover, as the results below indicate, KAAT1 and CAATCH1 exhibit striking differences in physiology and function. Parallel experiments conducted on the wild types and several sitedirected mutants of both KAAT1 and CAATCH1 cDNAs confirmed that they are functionally distinct transporters. 2 The functional characteristics of CAATCH1 expressed in Xenopus oocytes were unexpected and collectively did not conform to any known transport system, including KAAT1 or the excitable tissue transporters (18, 23, 33-35). Nutrient amino acid substrates for CAATCH1 elicited three categories of electrophysiological responses (Figs. 2-4): 1) net inward currents, typified by proline; 2) apparent net "outward" responses typified by methionine; and 3) a mixed net effect of elicited inward current plus apparent "outward" current, typified by threonine. Furthermore, the amino acid-elicited responses were modulated by Na ϩ , K ϩ , pH, and the transmembrane voltage but not by Cl Ϫ .
The first type of substrate, such as L-proline and its analogue L-pipecolate, elicited the greatest net inward currents of any test substrates in alkaline Na ϩ medium (Fig. 2, A and C). These currents were voltage-dependent over the range from Ϫ150 to ϩ30 mV (Fig. 3A).
The second type of substrate, such as methionine or leucine, blocked the CAATCH1-associated leakage current, thereby giving apparent "outwardly directed" current traces in Na ϩ medium (Fig. 2, A and C). The blockage of a CAATCH1-associated leakage current by these amino acids was characterized by a reduced slope of the current-voltage relation in the presence of substrate, compared with the control conditions. The CAATCH1 leakage current I-V relationship for methionine ( Fig. 3B) was obtained by subtracting I-V traces measured in the presence of this leakage-blocking substrate from those measured in its absence. Sonders et al. (10) used this method to reveal the cocaine blockage of the hDAT leakage current, which was independent of cocaine's competitive inhibition of dopamine carrier-mediated uptake via hDAT. Methionine-blocked CAATCH1 leakage currents in Na ϩ medium (Fig. 2, A and C) gave an outwardly rectifying I-V relationship (I CON Ϫ I MET ) with reversal potentials occurring between Ϫ10 and Ϫ25 mV (Fig. 3B).
The third type of amino acid, exemplified by threonine, elicited no current or small inward currents (Fig. 2, A and C), yielding an inverted U-shaped (convex) I-V relationship (Fig.  3A), since their CAATCH1-associated inward currents were balanced by their blockage of the CAATCH1-associated leakage current in Na ϩ medium (Fig. 2, A and C).
Complete replacement of extracellular Na ϩ with K ϩ changed the apparent substrate selectivity; thus, threonine (Fig. 2, B and D) elicited larger voltage-dependent inwardly rectifying currents than proline (Fig. 2, B and D; Fig. 3C). Furthermore, amino acids that blocked the leakage current in Na ϩ medium (Fig. 2C) generated net inward currents in K ϩ medium (Fig.   FIG. 2. Changes in amino acid selectivity of CAATCH1 as a function of Na ؉ or K ؉ . A, inward current in Na ϩ medium elicited by proline, meager inward current elicited by threonine, and block of inward current elicited by methionine (giving an apparent "outward" tracing). B, inward current in K ϩ medium elicited by threonine and moderate inward current elicited by proline. C, relative amino acid selectivity and current magnitudes in Na ϩ medium normalized to proline response. D, relative amino acid selectivity and current magnitudes in K ϩ normalized to threonine response. E, cation preferences for threonine-or prolineelicited CAATCH1 currents. Extracellular buffer Na ϩ and K ϩ salts were completely replaced by chloride salts of the indicated metal cations, choline, or NMG. In A and B, all recordings were made from the same representative cell. A small inward response to threonine in Na ϩ medium is shown; in other cells, threonine elicited the apparent "outward current" (block of inward leak), typified by methionine, as in A. Amino acids (500 M) were added to the superfusate buffer (pH 8.0) containing exclusively Na ϩ , K ϩ , or indicated cation; holding potential difference ϭ Ϫ60 mV. 2D). Methionine elicited only inward current in K ϩ medium, whereas it blocked the leakage current in Na ϩ medium. In K ϩ medium, test amino acids elicited I-V curves with strong inward current rectification from ϩ30 mV to at least -150 mV (Fig. 3C). Na ϩ and K ϩ were the favored activator cations; completely replacing all extracellular buffer Na ϩ and K ϩ with Rb ϩ , Li ϩ , NMG ϩ , or choline ϩ (chloride salts) attenuated amino acid-associated currents by 75-99% (Fig. 2E). Choline activated a small current with amino acid substrates, while NMG was virtually inert.
Threonine elicited small net inward currents (Ͻ10 nA) in some oocytes expressing CAATCH1 but elicited "outward" responses (i.e. CAATCH1-associated leakage current block) in other oocytes. This seemingly paradoxical phenomenon was resolved by analysis of threonine's unusual inverted U-shaped I-V relationship (Fig. 3A); this trace represented the combined effects of the inward threonine uptake-associated current and the simultaneous threonine inhibition of the CAATCH1-associated leakage current. At sufficiently negative voltages (e.g. Ϫ75 mV), current via the voltage-dependent transporter (uptake) pathway exceeded that of the exposed leakage current, resulting in a net inward current; however, at less negative voltages, the inhibition of leakage current approached or exceeded the transport-associated current, resulting in either a very small inward current or a net "outwardly directed" deflection. This behavior led to the complex I-V relationship of Fig.  3A. A similar phenomenon was reported for the action of dopamine on DAT and for the action of 5-HT on SERT (10,36).
The combined effects of methionine and proline are shown in Fig. 4A; increasing methionine concentrations blocked the constitutive CAATCH1 leakage current and inhibited proline-elicited inward transport-associated current in Na ϩ medium. Furthermore, I-V relationships (Fig. 4B) measured with various methionine/proline concentration ratios demonstrated a concentrationdependent inhibition of the net inward current while revealing the inhibited leakage current and its reversal potential. To eliminate the CAATCH1 leakage current from the total inward current obtained in the presence of proline, data were subsequently analyzed at the leakage reversal potential (i.e. Ϫ24 mV gave I leak ϭ 0 nA in Figs. 3B and 4, A-C). Under these conditions, methionine competitively inhibited the Na ϩ -activated proline-elicited component of net current (Fig. 4C) with an apparent K i ϭ 12 Ϯ 2 M methionine (calculated by Dixon analysis).
In both Na ϩ and K ϩ media, the kinetics of proline-and threonine-elicited transport-associated inward currents were fitted to the Michaelis-Menten equation, yielding hyperbolic kinetics that indicated single-site carrier behavior (data not shown). For the Na ϩ -activated proline current, K m ϭ 330 Ϯ 15 M, whereas for the Na ϩ activated threonine current, K m ϭ 35 Ϯ 4 M. For the K ϩ -activated proline current, K m ϭ 1900 Ϯ 270 M, and for the K ϩ -activated threonine current, K m ϭ 235 Ϯ 5 M.
Current-voltage relationships were determined for threonine currents activated at increasing concentrations of K ϩ and for proline currents activated by increasing concentrations of Na ϩ . Activation data were then fitted to the Hill equation by nonlinear regression, as illustrated in Fig. 5A. The apparent Hill coefficient, , was ϳ2 for both K ϩ and Na ϩ , implying an activation coupling ratio of 2 K ϩ :1 threonine and 2 Na ϩ :1 proline. CAATCH1 does not require any Cl Ϫ for amino acid-elicited inward current activity (Fig. 5B). Complete replacement of chloride with gluconate in Na ϩ -containing medium elicited proline-associated currents that were not significantly different (p Ͼ 0.05) at 100 mM Cl Ϫ (100 Ϯ 6%) from currents at 0 mM Cl Ϫ (92 Ϯ 9%). This phenomenon contrasts with the requirement for extracellular Cl Ϫ ion observed in cation-activated monoamine cotransporters and in KAAT1 (8 -10, 12, 13, 18, 23, 29, 35, 37). Although unaffected by Cl Ϫ , cation-activated amino acidelicited CAATCH1 inward currents were activated as a function of pH (or OH Ϫ concentration) (Fig. 5C). In CAATCH1expressing oocytes the maximal pH was 9.5 (Fig. 5C), mirroring the in vivo alkalinity of M. sexta midgut (38). The slopes (data not shown) of the difference IV relations increased as a function of pH, in accordance with the increased transport-associated current measured at the fixed holding voltage of Ϫ60 mV (Fig. 5C).
L-Proline in Na ϩ medium elicited an inward current that was accompanied by an abrupt acid shift in the unstirred layer surrounding the CAATCH1-expressing oocytes (Fig. 6). Subsequent removal of L-proline abruptly shifted the pH back in the alkaline direction along with arrest of the inward current. The pH shift represented either OH Ϫ influx or H ϩ efflux occurring concurrently with Na ϩ -dependent proline-elicited inward current. This effect of CAATCH1 activity on pH contrasts with the FIG. 3. Changes in amino acid-dependent current-voltage relations as a function of Na ؉ or K ؉ . All recordings were made from the same representative cell. Voltage ramps (ϩ30 to Ϫ150 mV during 5 s) were applied during constant superfusion of each amino acid (500 M) in either Na ϩ or K ϩ medium. A, proline-or threonine-elicited currents in Na ϩ medium. The threonine-elicited CAATCH1 current was slightly greater than the concomitant block of the constitutive CAATCH1 leakage current, yielding a convex shaped I-V relationship due to the additive effects of the inwardly rectifying transport-associated current with the inverted outwardly rectifying leakage current. B, leakage block I-V curve elicited by methionine in Na ϩ . Values represent CAATCH1 constitutive leakage current inhibited in the presence of the amino acid; the CAATCH1 reversal potential of this particular example was Ϫ15 mV. C, proline-or threonine-elicited currents in K ϩ medium. pH relationships of other eukaryotic transporters, which are energized by pH gradients or co-activated by H ϩ inward flux (36).
Current-voltage relationships were measured at six extracellular pH values from pH 7.0 to 9.4 in the absence of external monovalent cations (replacement by NMG ϩ ). A linear fit of the reversal potentials as a function of pH (data not shown) gave a slope of Ϫ11 Ϯ 0.2 mV/pH unit. This value is less than that predicted for a perfect Nernst potential for H ϩ (or OH Ϫ ), indicating that other ions (probably intracellular K ϩ or Na ϩ ) carry a greater proportion of the current through the leakage pathway.
CAATCH1 cation-associated current-voltage relationships, independent of the substrate-associated currents, were observed in the absence of leakage blocking amino acids (Fig. 7A). Here, the rightward shift in the reversal potential and the positive slopes indicates a conductance for several cations, with a preference of Li ϩ Ͼ Na ϩ Ͼ K ϩ . However, when methionine was present (Fig. 7B), the negative slope (inverse leakage current) of the difference I-V relationship in Na ϩ unmasked the CAATCH1 leakage current that is apparently constitutively present in the absence of any test amino acid. The inwardly rectifying positive slopes of the difference curves in Li ϩ and K ϩ (Fig. 7B) indicate that these cations can also activate methionine-associated carrier currents.
In addition to its rheogenic properties, CAATCH1 also catalyzed carrier-mediated uptake of 3 H-labeled amino acid substrates (25 M). In intact oocytes expressing CAATCH1, uptake values obtained in medium with Na ϩ as the only alkali cation, under non-voltage-clamped conditions and corrected for nonspecific uptake measured in water-injected oocytes, were as FIG. 4. Methionine block of CAATCH1 constitutive leakage current, and methionine inhibition of proline transport-associated inward current. A, effects of methionine and proline on CAATCH1 currents in Na ϩ medium. Proline alone (P; 500 M) elicited an inward transport-associated current, whereas methionine alone (M; 500 M) blocked the constitutive CAATCH1 leakage current. The proline-elicited steady-state inward current was increasingly inhibited by 0.005-500 M methionine. Blanked voltage ramp recordings are marked by triangles. Membrane voltage was clamped at Ϫ60 mV. B, CAATCH1 current-voltage relations with proline and methionine combinations in Na ϩ medium. I-V relations were derived from voltage ramp commands as in A. For clarity, a limited number of proline and/or methionine I-V curves are shown. For each steady-state I-V curve except Met only (leak), the data were plotted according to the convention for transport-associated currents, i.e. corrected for base line by digital subtraction of control base-line ramp currents (measured immediately prior to the application of amino acids) from the ramp current measured during the amino acid application. Met only (leak) shows the leakage I-V relation (dashed line) in its conventional form, in which current in the presence of methionine was subtracted from the base-line control current. Met only (inverse leak) represents the data (dotted line) plotted in the form normally used for transport-associated currents. C, methionine inhibition of inward proline-induced CAATCH1 current. To isolate net transport-associated currents (at leakage current ϭ 0 nA), measurements were obtained from the current records during the voltage ramps (see A and B) at a voltage representing the reversal potential of the leakage current (i.e. Ϫ24 mV in this example). These data represent methionine inhibition of only the proline-associated net current in Na ϩ medium.

FIG. 5. Ion activation kinetics of amino acid-elicited CAATCH1 currents.
A, Hill plot of K ϩ -activated threonine current. Empirical data were fitted to the Hill equation by nonlinear regression. The Hill coefficient, , was 1.8 with K Hill 0.5 ϭ 92 mM and I max ϭ 224 nA at potential difference ϭ Ϫ60 mV. Similar data were obtained for Na ϩ -activated proline currents, which yielded an apparent Hill coefficient of ϭ 1.5 Ϯ 0.1, with K Hill 0.5 ϭ 30.1 Ϯ 0.9 mM and I max ϭ 85 Ϯ 5 nA at potential difference ϭ Ϫ60 mV. Similar curves were obtained at additional voltages ranging from Ϫ60 to Ϫ150 mV. B, lack of Cl Ϫ effect on prolineelicited current in Na ϩ medium. C, effect of extracellular pH on prolineelicited (500 M) CAATCH1 current in oocytes. Inward current values were obtained by normalizing to responses in medium buffered with TAPS/NMG at pH 8.0; media were buffered with HEPES/NMG (pH range 7.0 -8.0) or TAPS/NMG (pH range 8.0 -10.0). follows (pmol/min/oocyte; mean Ϯ S.E.): L-proline, 2.10 Ϯ 0.20; L-threonine, 1.98 Ϯ 0.30; L-alanine, 0.82 Ϯ 0.30; and L-methionine, 0.2 Ϯ 0.10. Repeated attempts to measure uptake in medium with K ϩ as the only alkali cation varied quite widely from batch to batch of injected oocytes, yielding error values often about Ϯ 100%. However, radiolabeled amino acid uptake values in the Na ϩ -free K ϩ medium were generally greater than water-injected control oocytes but less than uptake in K ϩ -free Na ϩ medium. The inability to derive meaningful measurements under Na ϩ -free K ϩ experimental conditions is probably due to a lack of significant driving force for transport (i.e.

DISCUSSION
CAATCH1 is a unique clone that shares some structural and functional characteristics with Na ϩ ,Cl Ϫ -coupled neurotransmitter transporters of excitable tissue but also shares characteristics of epithelial nutrient amino acid transport systems. The unanticipated characteristics of CAATCH1 included Cl Ϫ insensitivity, inward currents that are elicited by nutrient amino acids and activated by either K ϩ or Na ϩ , substrate preference that depends on the activator cation (e.g. threonine with K ϩ but proline with Na ϩ ), alkaline pH activation increasing to pH 9.5, voltage dependence, and, notably, the inverted U-shaped current-voltage relationship of a ligand-inhibited "apparently outward" leakage current initially ascribed to neurotransmitter transporters such as DAT (8 -10). The DAT, NET, SERT, GABA, GAT1, EAATx, GLAST, GLT, PROT, and GlyTx transporters each mediate uptake of solute (primarily neuroactive amines) via a carrier function, yet they also exhibit substrate-independent ion leakage channels that can be inhibited by pharmacological agents in some instances (1-5, 8 -13, 29, 35-37, 40 -43). Na ϩ -dependent leakage currents have also been reported in nonexcitable tissues (e.g. a thyroid Na ϩ /I Ϫ uptake system (15), myo-inositol (16), and Na ϩ /glucose (SGLT1) transporters (3,17) and ASCT-x Na ϩ , Cl Ϫ dependent transporters broadly selective for both neutral and acid amino acids (2,5)). The mammalian amine and amino acid transporters are strictly dependent on Cl Ϫ , unlike CAATCH1 (Fig. 5). Related insect transporters, dSERT of Drosophila (48) and a Manduca GABA transporter (30), each retain about 50% of Na ϩ -dependent serotonin or GABA uptake when all Cl Ϫ is replaced by gluconate.
KAAT1 and CAATCH1 appear to be related, but they are different proteins with different functions. Although they are 90% identical, they diverge at 62 positions distributed among many sites within the overall structure (Fig. 1), as described under "Results." It is possible to conceive of complex patterns of alternate splicing of transcripts derived from a single gene to account for these differences, although RNA editing of a single transcript or a gene duplication could also account for the variations. Both clones display the property of voltage-dependent, substrate-elicited currents activated by K ϩ at alkaline pH (Figs. 2, 3, and 5) (18), and they both display currents elicited FIG. 6. Concomitant shifts in Na ؉ -dependent proline-elicited inward current and pH of extracellular unstirred layer. A, inward CAATCH1 current elicited by 500 M proline in Na ϩ medium. L-Proline in Na ϩ medium (pH 8.410 Ϯ 0.003) was slowly superfused by a peristaltic pump. A micro-pH electrode was positioned in the extracellular medium 50 m from the surface of a CAATCH1-expressing oocyte that was impaled with a two-electrode voltage clamp. Current and extracellular pH measurements were obtained simultaneously. Gaps appear where deflections resulting from applied voltage ramps were removed. B, acute pH shifts in extracellular medium were coincident with Na ϩ /proline-elicited inward current. Proline applied at t ϭ 0 resulted in an abrupt extracellular medium acidification pH shift representing either an H ϩ efflux or OH Ϫ influx coincident with Na ϩ / proline-elicited inward current. An abrupt alkaline pH shift occurred upon removal of proline, coincident with cessation of Na ϩ /proline-elicited current. by Na ϩ in the absence of substrates (Fig. 7) (39), but the substrate selectivity and electrophysiological properties of the two transporters are otherwise quite different. For example, in addition to the list of CAATCH1's unique properties summarized above, the CAATCH1 leakage current is modulated by micromolar methionine (Figs. 2-4), whereas a KAAT1 constitutive current is not inhibited by any known agent (34,39). Moreover, our initial studies of single site and double site mutations show striking functional differences between CAATCH1 and KAAT1. 2 With an apparent reversal potential of approximately Ϫ20 mV in Na ϩ medium, the observed CAATCH1 leakage phenomenon is probably a complex event, with the actual ion(s) responsible for it being unknown at present. The CAATCH1 conductance as well as its net inward holding currents measured in the presence of various single alkali cation species are greater than values obtained when NMG ϩ replaces all alkali cations (Fig. 7). This suggests that the CAATCH1 leakage pathway could be conductive to at least Li ϩ , K ϩ , and Na ϩ . The presence of a high Li ϩ conductance is consistent with observations for other members of the Na ϩ -dependent neurotransmitter transporter family. Furthermore, in alkali cation-free NMG ϩ medium, CAATCH1 holding currents increased as a function of extracellular [H ϩ ], with the I-V slopes increasing at lower pH (giving reversal potentials at Ϫ11 mV/pH unit), suggesting that protons may also permeate the leakage pathway (see "Results"). The lack of Cl Ϫ activation and the dependence on alkaline pH (Figs. 5 and 6) raises the possibility that OH Ϫ may serve as an activator anion for CAATCH1. It is difficult to study the ionic dependence of the ligand-inhibitable leakage pathway in CAATCH1 because it is detectable only in the presence of Na ϩ (Fig. 2, C and D). Taken together, our observations suggest that H ϩ influx and/or OH Ϫ efflux may contribute to the net leakage current in the absence of Na ϩ but that Na ϩ is the major contributor when it is present.
The current-voltage, pH, substrate concentration, and ion concentration data suggest that CAATCH1 may exist in one or more states, as has been postulated for amine or amino acid neurotransmitter transporters (3)(4)(5)8). At least two states could exist that are regulated by the available cation and amino acid substrates: 1) a Na ϩ -dependent state having a constitutive leakage pathway that is inhibited by certain amino acids, such as methionine, but that can generate inward transport-associated currents in the presence of a narrow spectrum of amino acids (e.g. proline) (some amino acids, such as threonine, may both inhibit the leakage current and produce transport-associated currents) and 2) a K ϩ -dependent state in which transportassociated currents are elicited by a broad spectrum of neutral amino acids, such as threonine or methionine, whose predominant effects in the presence of Na ϩ are the inhibition of the leakage currents.
The leakage current and its modulation by amino acid ligands are not predicted by traditional models but are in accord with recent observations in other transporters (13, 14, 40, 44 -46). Our data are consistent with a model in which 1) the test amino acids bind to a common receptor site on CAATCH1; 2) the avid binding of most test amino acids (except proline) prevents inward leakage current in Na ϩ medium, where the predominant effect of methionine is to expose the CAATCH1associated leakage conductance; and 3) the binding affinity for most amino acids is reduced in K ϩ medium, thereby allowing the transport cycle to be completed with concomitant generation of inward carrier-associated currents.
CAATCH1 properties can account for several characteristics of amino acid transport observed in insect larval epithelium (38). The characteristics of CAATCH1-dependent radiolabeled amino acid uptake by oocytes (see "Results") are consistent with those found in radiotracer uptake studies in isolated membrane vesicles from Manduca midgut (38). This earlier vesicle work demonstrated multiple, physiologically separate uptake "systems" for alkali cation-activated amino acid co-transport in Manduca epithelial cells (47). The K ϩ -activated, leucine-preferring KAAT1 transporter was also previously expressioncloned from Manduca RNA (18).
In conclusion, the amino acid carrier and apparent ion leakage properties of CAATCH1, previously attributed to the organic solute transporters of excitable tissues, provide insights into the means of electrogenic amino acid uptake in the highly alkaline (pH Ͼ 10) caterpillar midgut. The alkalinity is maintained by an H ϩ V-ATPase that generates an approximately Ϫ240-mV transmembrane voltage in conjunction with a putative K ϩ /2H ϩ antiporter (38). The alkaline milieu breaks down dietary tannins that block amino acid absorption, while vesicle studies show that the main function of the large voltage is to drive K ϩ and amino acids into the epithelial cells via transport systems (38). The characteristics of CAATCH1 described here pave the way for a detailed electrophysiological analysis of CAATCH1's transport solute/current stoichiometry and presteady state kinetics.