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A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea

1999; Springer Nature; Volume: 18; Issue: 20 Linguagem: Inglês

10.1093/emboj/18.20.5505

1460-2075

Bettina Bölter,

Plant nutrient uptake and metabolism

Article15 October 1999free access A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea Bettina Bölter Bettina Bölter Botanisches Institut, Universität Kiel, D-24118 Kiel, Germany Search for more papers by this author Jürgen Soll Corresponding Author Jürgen Soll Botanisches Institut, Universität Kiel, D-24118 Kiel, Germany Search for more papers by this author Kerstin Hill Kerstin Hill Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Roland Hemmler Roland Hemmler Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Richard Wagner Richard Wagner Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Bettina Bölter Bettina Bölter Botanisches Institut, Universität Kiel, D-24118 Kiel, Germany Search for more papers by this author Jürgen Soll Corresponding Author Jürgen Soll Botanisches Institut, Universität Kiel, D-24118 Kiel, Germany Search for more papers by this author Kerstin Hill Kerstin Hill Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Roland Hemmler Roland Hemmler Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Richard Wagner Richard Wagner Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany Search for more papers by this author Author Information Bettina Bölter1, Jürgen Soll 1, Kerstin Hill2, Roland Hemmler2 and Richard Wagner2 1Botanisches Institut, Universität Kiel, D-24118 Kiel, Germany 2Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany ‡B.Bölter and R.Hemmler contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5505-5516https://doi.org/10.1093/emboj/18.20.5505 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphorylated carbohydrates are the main photoassimilated export products from chloroplasts that support the energy household and metabolism of the plant cell. Channels formed by the chloroplastic outer envelope protein OEP21 selectively facilitate the translocation of triosephosphate, 3-phosphoglycerate and phosphate, central intermediates in the source–sink relationship between the chloroplast and the cytosol. The anion selectivity and asymmetric transport properties of OEP21 are modulated by the ratio between ATP and triosephosphates, 3-phosphoglycerate and phosphate in the intermembrane space. Conditions that lead to export of triosephosphate from chloroplasts, i.e. photosynthesis, result in outward-rectifying OEP21 channels, while a high ATP to triosephosphate ratio, e.g. dark metabolism, leads to inward-rectifying OEP21 channels with a less pronounced anion selectivity. We conclude that solute exchange between plastids and cytosol can already be regulated at the level of the organellar outer membrane. Introduction Members of the plastid organelle family carry out vital biosynthetic functions in every plant organ. Chloroplasts, chlorophyll-containing plastids, carry out photosynthesis, which converts atmospheric carbon dioxide to carbohydrates such as triosephosphate, starch and others. These and further biosynthetic pathway products and intermediates are exchanged continuously with the parent cell with the assistance of specific carrier proteins localized in the plastidic inner envelope (Flügge, 1998) and solute channels located in the chloroplastic outer envelope. While the inner envelope transport proteins, e.g. the triosephosphate/phosphate translocator (TPT), the dicarboxylic acid translocator or the hexose phosphate carrier, show a distinct substrate selectivity and specificity, it is not clear to what extent transport through the outer membrane channels is selective and regulated. Furthermore, it is not known how many different channels are present and are required in the outer membrane for plastid function. In mitochondria, a major solute channel with high conductance is represented by the voltage-dependent anion channel (VDAC) (Zoratti and Szabo, 1995; Kinnally et al., 1996). Although evidence exists that besides the porin-like VDAC channel additional high conductance channels could be present in the mitochondrial outer membrane, only a second VDAC-like protein has been identified (Benz, 1994; Szabo et al., 1995). In Gram-negative bacteria, however, several different types of high conductance channels exist in the outer membrane (Nikaido, 1993). (i) So-called porins form water-filled pores that allow the downhill diffusion of solutes, provided that the size of the solutes does not exceed the exclusion limit (∼600 Da) of the channel pore (OmpF) (Schirmer et al., 1995). Modulation of these channels by ATP and other effectors has been reported for some of these porins, which casts doubt on the concept of a generally open diffusion pore (Rudel et al., 1996; Delcour, 1997; Iyer and Delcour, 1997; Samartzidou and Delcour, 1998, 1999). (ii) Porin-like channels, e.g. LamB from Escherichia coli, carry specific sites through which selective diffusion processes are facilitated (Keller et al., 1994; Schirmer et al., 1995). (iii) Ligand-gated pores, e.g. E.coli ferric enterobactin channels (FePA), provide energy-dependent uptake of nutrients into bacteria (Rutz et al., 1992; Jiang et al., 1997). The ancestral relationship of mitochondria and plastids to Gram-negative bacteria (Margulis, 1970; Gray, 1993; Martin and Müller, 1998) suggests the presence of multiple channel proteins in the organellar outer membrane. In pea chloroplast outer membranes, three channel proteins have been identified and functionally characterized thus far. The preprotein-conducting channel with prokaryotic ancestors is formed by Toc75 (Hinnah et al., 1997; Bölter et al., 1998). The outer envelope protein of 16 kDa (OEP16) forms a cation-selective highly conductive channel with a strong bias for amino acids and amines (Pohlmeyer et al., 1997). The channel characteristics of the third channel protein identified, OEP24 (Pohlmeyer et al., 1998), closely resemble those described for general diffusion pores (Benz, 1994). The gating properties, i.e. opening and closing of different channels, might be co-ordinated to reflect the metabolic needs of the communicating partners, e.g. chloroplast and cytosol. Little is known about how modulation of outer membrane channels is achieved in vivo, because a substantial membrane potential, as simulated in vitro by a voltage gate, is not likely to exist across the outer membranes in situ (for a review, see Klebba and Newton, 1998). Here we describe the chloroplastic outer envelope protein from pea, OEP21. Reconstituted OEP21 protein forms a voltage-dependent, anion-selective channel. Of note is the asymmetric current–voltage relationship observed, as well as the tight modulation of the OEP21 channel activity by nucleotides and carbon intermediates of photosynthesis from the intermembrane space between the inner and outer plastid membrane. We conclude that the transport from and into plastids can already be regulated at the level of the outer membrane. Results Candidate outer envelope channel proteins should possess most of the characteristics of known channel proteins: (i) they are relatively abundant, because plastids are involved in many high 'throughput' biosynthetic pathways; (ii) many channel proteins are deeply embedded in the membrane and are resistant to proteolysis, e.g. the mitochondrial VDAC or the chloroplastidic OEP24, OEP16 and Toc75; and (iii) most channel proteins have a neutral or alkaline isoelectric point, e.g. VDAC, OMPF, OEP16 and OEP24. An unknown outer envelope polypeptide from pea with an apparent mol. wt of 21 kDa was found to be abundant on SDS–PAGE (Figure 1B) and exhibited an alkaline isoelectric point. We therefore decided to analyse this protein in detail. Figure 1.Localization and distribution of OEP21 in pea. (A) Amino acid sequence as deduced from the clones peacOEP21 and peagOEP21. The N-terminal and internal amino acid sequences of OEP21 overlapped and are shown underlined. (B) Polypeptide composition of purified outer envelope membranes from pea (lane 1) and recombinant OEP21 (lanes 2–5). Recombinant OEP21 was recovered from insoluble inclusion bodies (lane 2) and purified by cation-exchange chromatography (lane 3). The eluate (lane 3) was fractionated further by size exclusion chromatography: lane 4 shows the last collected fraction that contains a low molecular weight protein; lane 5, OEP21 used for reconstitution. Numbers on the left indicate molecular weight standards in kDa. (C) Immunoblot analysis of the presence of OEP21 in the outer envelope (OE), inner envelope (IE) and thylakoids (THY) from pea chloroplasts (10 μg of protein were loaded per lane), pea etioplasts, potato mitochondria or total membrane proteins from leaf, shoot or roots from pea (100 μg of protein loaded per lane). PIS, pre-immune serum; α-OEP21, antiserum to OEP21. (D) Purified outer envelope membranes were either not treated (−) or treated (+) with the protease thermolysin (Th), extracted with Na2CO3 at pH 11, 1 M NaCl or 4 M urea and separated into a soluble (S) and insoluble membrane fraction (M). An immunoblot is shown using an OEP21 antiserum. The arrowheads indicate two proteolytic fragments of OEP21. (E) Purified intact pea chloroplasts (equivalent to 20 μg of chlorophyll) were either not treated or treated with thermolysin. Total chloroplast membrane proteins were separated by SDS–PAGE and analysed for the presence of the different envelope proteins by immunoblot. (F) Immunogold labelling of ultra thin sections from pea leaves using the OEP21 antiserum. An overview is shown on the left. The right upper and middle panels represent independent labelling events of pea chloroplasts. The lower right panel is an enlargement of the overview indicated by the arrowhead. The scale bar represents 100 nm for the enlargements and 360 nm for the overview. Download figure Download PowerPoint N-terminal and internal peptide sequences with overlapping sequence information (Figure 1A) were obtained for the 21 kDa protein after SDS–PAGE separation of purified outer envelope membranes. The N-terminal protein sequence started with the amino acid glutamine, indicating that either the start methionine had been removed after translation, as detected frequently in a eukaryotic system (Walker and Bradshaw, 1999), or that the 21 kDa polypeptide represents only a proteolytic fragment of a larger protein. Digoxygenin-labelled oligonucleotides were used to isolate the corresponding cDNA clone from a pea expression library. Both peptide sequences were found in the deduced open reading frame (ORF), thus demonstrating that the cDNA clone peacOEP21 codes for the 21 kDa protein (Figure 1A). However, the deduced amino acid sequence of peacOEP21 lacked the start methionine and the first triplet coded for glutamic acid instead of glutamine in the protein sequence. A new homologous screening of the pea cDNA library did not result in a different isolate. We therefore decided to screen a pea genomic DNA library. The deduced ORF of the genomic clone peagOEP21 contained the missing start methionine, but had no further coding sequence information compared with peacOEP21. PeagOEP21 contained a stop codon 36 bp upstream of the putative start ATG codon. From these data, we conclude that the outer envelope 21 kDa polypeptide represents a full-length protein, from which methionine has been removed after translation. A protein of 177 amino acids, a calculated mol. wt of 20.8 kDa and an isoelectric point of 9.6 can be deduced from the coding sequence (Figure 1A) which was deposited in the DDBJ/EMBL/GenBank (accession No. AJ009987). We named the protein OEP21. Protein sequencing of OEP21 from two independent outer envelope preparations (Figure 1B) indicated glutamine as the N-terminal amino acid, while the ORFs of both peacOEP21 and peagOEP21 code for glutamic acid. It remains to be established whether this is due to a post-translational modification or to an in vitro artefact during the isolation of the OEP21 polypeptide. A database search revealed no significant homologies to other proteins, except to expressed sequence tags from rice and corn (accession Nos D49096 and AJ006545, respectively), indicating the presence of OEP21 in both monocotyledonous and dicotyledonous plants. Sequence analysis reveals that OEP21 has an unusually high content of polar and charged amino acids (51%) for a membrane protein. Indeed, a computer algorithm that is also able to predict membrane proteins, e.g. TopPredII (von Heijne, 1992), indicated that OEP21 is likely to be a soluble protein. To establish further its chloroplast origin, immunolocalization and protein translocation experiments were conducted. An antiserum raised against the recombinant protein recognized one protein in the chloroplastic outer envelope. Little or no cross-reaction occurred with inner envelopes or thylakoids and mitochondria (Figure 1C), respectively. OEP21 is present in chloroplasts, etioplasts and non-green plastids from roots, demonstrating its presence in different plastid types (Figure 1C). OEP21 is resistant to extraction at pH 11.5, with high salt and 4 M urea, respectively, thus behaving like an integral membrane protein (Fujiki et al., 1982; Figure 1D). Treatment of right-side-out outer envelope vesicles with the protease thermolysin results in two distinct proteolytic fragments of 14 and 10 kDa apparent mol. wt (Figure 1D). Both fragments are resistant to extraction at pH 11.5. When intact chloroplasts were treated with the protease thermolysin, the outer envelope proteins Toc160, Toc34 and OEP21 were proteolytically cleaved. The proteolytic fragments obtained from OEP21 in intact chloroplasts were of similar size to those in isolated outer membranes. The thermolysin-resistant protein Toc75 and the inner envelope protein Tic110 were not degraded by thermolysin in intact chloroplasts (Figure 1E). Together, these data indicate that OEP21 exposes a protease-sensitive loop at the cytoplasmic face of the outer envelope membrane (see also below). Immunogold labelling of ultra thin sections from pea leaves (n >100) also supports the chloroplast origin of OEP21 and its localization in the outer envelope (Figure 1F). Radiolabelled OEP21 translation product was found to insert into intact chloroplasts that had either not been treated or been treated with protease to remove surface-exposed receptor components that initiate translocation of precursor proteins (Figure 2A). The OEP21 translation product inserted slightly more effectively into protease-pretreated chloroplasts, perhaps because the accessible lipid membrane surface increases due to the removal of large exposed polypeptide epitopes. Insertion of proteins without a cleavable transit sequence is also independent of ATP (Figure 2C). In contrast, the precursor of the stromal protein SSU (small subunit of ribulose-1,5-bisphosphatecarboxylase, pSSU) requires the hydrolysis of ATP for import (>100 μM) and the presence of protease-sensitive presequence receptors at the organellar surface (Figure 2B). From the results presented in Figures 1 and 2, we conclude that OEP21 is indeed localized in the outer envelope of pea chloroplasts. Figure 2.Comparison of OEP21 and pSSU translocation into chloroplasts. Intact pea chloroplasts were incubated with 35S-labelled OEP21 (A and C) or pSSU (B) translation product (TL) for 10 min at 25°C. Chloroplasts were either not treated or treated with the protease thermolysin prior to the translocation reaction as indicated on the top of the figure. (A) Chloroplasts were recovered after import, washed and either not treated or treated with Na2CO3 at pH 11.5 or 1 M NaCl as indicated and separated into soluble (S) and insoluble membrane (M) fractions. (B and C) Insertion of pSSU and OEP21 translation product in the absence (−) or presence (+) of 2 mM ATP. TL, translation product, 10% of which was added to an import reaction. A fluorogram is shown. Download figure Download PowerPoint Expression and structural analysis of OEP21 In order to characterize the functional properties of OEP21, the pea protein was heterologously expressed in E.coli cells. The protein was recovered from insoluble inclusion bodies, denatured in 6 M guanidine–HCl and purified further by cation exchange chromatography and size exclusion chromatography. The isolation protocol resulted in OEP21 purified to apparent homogeneity (Figure 1B, lanes 2–5). The circular dichroism (CD) spectra of the reconstituted heterologously expressed OEP21 protein show that β-sheets are the most frequent secondary structure element (Figure 3A). The CD data also allow calculation of the relative content of secondary structures (α = 0.11; β = 0.46; P2 = 0.23; other = 0.20) (Figure 3B) Pohlmeyer et al., 1997, 1998; Hill et al., 1998). These results are in line with the secondary structure predictions by the PHD or PSD programs (Pattern Structure Database), which both predict an α-helix content 40%. Furthermore, available computer algorithms do not indicate the presence of a transmembrane α-helix in OEP21 (Gilbert, 1992; Rost and Sander, 1993; Rost et al., 1994) (Figure 3C). From the above predictions, we obtained the putative secondary structure and hydropathy profile as outlined in Figure 3C. According to these results, OEP21 contains eight amphipatic β-strands spanning the membrane and a hydrophilic loop from about G107–G108 to approximately G124–G125 carrying four positive charges and two adjacent positive charges (K104, K106; see Figure 1A). The putative secondary structure resembles that of OMP21 from Comamonas acidovorans and other similar proteins that are characterized by eight transmembrane β-strands, large exoplasmic loops and a C-terminal domain in the periplasmic (intermembrane) space. Some of these proteins are proposed to form membrane pores (Baldermann et al., 1998). In the hydrophilic C-terminal part of the protein, we localized a potential ATP-binding site of the FX4K type, which has a rectifying role in potassium-selective channels (D.B.McIntosh et al., 1996; Drain et al., 1998). Support for functionality of this motif comes from photoaffinity labelling with azido ATP (Figure 3D, which demonstrates that OEP21 binds ATP. In order to identify proteins in close physical proximity to OEP21, purified outer envelope membranes were incubated with different chemical cross-linking reagents. No partner proteins could be detected except for the formation of homo-oligomeric states of OEP21. Immunoblotting and co-immunoprecipitation experiments indicated that the dimeric form was most prominent, but trimers were also detectable at higher cross-linker concentrations (Figure 3E). How this correlates with OEP21 function remains to be established. Figure 3.Structural analysis of OEP21. (A) CD spectra of OEP21 reconstituted into liposomes. CD spectra were recorded between 180 and 250 nm on a Jasco 600 CD spectropolarimeter, as described in Materials and methods. (B) Relative (rel.) abundance of OEP21 secondary structure reconstituted into liposomes, according to Sreerama and Woody (1993, 1994). P-2, disturbed helix in the presence of two prolines. (C) Hydropathy plot (Gilbert, 1992) predicted secondary structure (Rost and Sander, 1993) of the deduced amino acid sequence of OEP21. β-sheets are shown as arrows, putative helices as cylinders. (D) Affinity labelling of outer envelope membranes by [32P]N3-ATP in 254 nm UV light. Lane 1 shows total labelling of outer envelope proteins; lanes 2 and 3 show immunoprecipitations using OEP21 antiserum or pre-immune serum, respectively. A fluorogram is shown. (E) Outer envelope membranes (10 μg of protein) were incubated with the chemical cross-linker BS3 and analysed for the formation of OEP21 oligomers after SDS–PAGE by immunoblotting (left panel). Numbers on the left indicate the position of molecular weight markers in kDa. The right panel shows an immunoprecipitation in the absence or presence of α-OEP21 as indicated after cross-linking followed by immunoblotting and incubation with α-OEP21. Download figure Download PowerPoint OEP21 forms an intrinsically rectifying anion channel with subconductant states The data so far strongly supported our idea that OEP21 may form a solute channel. The purified protein was reconstituted into liposomes as described in detail previously (Pohlmeyer et al., 1997, 1998). After fusion of OEP21 liposomes with planar lipid bilayers, voltage-dependent single channel currents could be well resolved, provided measurements were performed in solutions with ionic strength ≥250 mM. Single channel activity was observed at both positive and negative membrane potentials (Figure 4A). The OEP21 channel displayed complex gating with multiple open channel amplitudes (Figure 4B). Direct transitions between the different open channel amplitudes were observed, indicating that these amplitudes represent subconductant states of a single channel rather than simultaneous openings or closures of independent channels with various amplitudes and rise times of gating 5) in symmetric 1 M NaCl (as deduced from Figure 4C). Frequent subconductance levels were observed at Λ1 = 430 ± 20 pS (n = 5) and Λ2 = 280 ± 20 pS (n = 5). In symmetric 250 mM NaCl, the highest conductance of the OEP 21 channel showed a value of = 350 ± 10 pS (n >20). The current–voltage relationship of the open single channel was asymmetric, even in symmetric buffers on both sides of the membrane (Figure 4C) as observed in >10 sets of independent bilayer experiments. This shows that the OEP21 channel is intrinsically rectifying, probably due to interactions of the permeating ions with the channel pore, which presumably exhibits an asymmetric potential profile along the channel pore (see also below). Moreover, the applied fusion technique yielded OEP21 channels incorporated into the bilayer membrane with a single preferential direction, otherwise the asymmetric current–voltage relationship (Figure 4C) could not have been observed. When a voltage gate of 60 s was applied across an OEP21-containing bilayer, the averaged mean currents passing the OEP21 channel decreased with increasing membrane potentials (Figure 4D). This decrease was more pronounced at negative potentials than at positive potentials. The data from Figure 4C and D have been obtained from the same bilayer, therefore they may be used to calculate the mean open probability (averaged for all sublevels) of the OEP21 channel at different membrane potentials (Figure 4E). Obviously, the open probability of the recombinant reconstituted channel is high (Popen >0.2) only in a narrow range of membrane potentials from Vm = 0 mV to Vm = +50 mV, while reconstituted envelope membranes contain open OEP21 channels at 0 mV (see below). In asymmetric buffers (2 M KCl/250 mM KCl cis/trans), the OEP21 channel revealed a reversal potential of Erev = −18.5 ± 2.8 mV (ECl− = −5 mV, EK+ = +35 mV) (Figure 4F). These results demonstrate that OEP21 forms an anion-selective channel. The lower conductance states of the OEP21 channel revealed a slightly higher selectivity for cations over anions (Figure 4F). The conductance (Λ1) with 40% of the fully open channel amplitude revealed a reversal potential of Erev = −28 ± 3.6 mV. Conversion of the reversal potential into relative permeabilities by the Goldman–Hodgkin–Katz (GHK) approach yielded the permeability ratios of PCl−/PK+ ≅ 3:1 and PCl−/PK+ ≅ 5:1, respectively. From the single channel conductance, we can estimate the approximate diameter of the aqueous OEP21 channel as dchannel ≅ 2.1 nm (Hille, 1968), taking into account that the conductivity of the electrolyte solution within the pore is ∼5× lower than in the bulk solution (Smart et al., 1997). Figure 4.Electrophysiological properties of purified recombinant OEP21. (A) Current recordings from a bilayer containing a single copy of an active channel at a holding potential Vh = −60 mV (upper) and Vh = +80 mV (middle). Lower panel, current recording and expanded view from the bilayer containing a single copy of an active channel at a holding potential of Vh = +80 mV shown in the middle panel. The cis and trans chamber contained 1 M NaCl, 10 mM MOPS–Tris pH 7.0. (B) All point amplitude histogram of the current trace. (C) Current–voltage relationship of the single channel deduced from single channel currents of the main conductance state in symmetric solutions of 1 M NaCl, 10 mM MOPS–Tris pH 7.0 in both compartments. Data points are an average of n = 5 independent bilayers. Error bars represent standard deviation of the data. (D) Mean current histogram in a bilayer containing a single copy of an active channel during an applied voltage gate of 60 s duration with different amplitudes (symmetric buffer 1 M NaCl, 10 mM MOPS–Tris pH 7.0). (E) Calculated mean open probability averaged for all open channel amplitudes. Data were calculated by dividing the averaged open channel currents measured in (D) by the maximum possible currents of the open main conductance state (B). Only mean current and open channel current data from the same bilayer were used to calculate Popen. Data points are an average of n = 3 independent bilayers. Error bars represent standard deviation of the data. (F) Current recording from a bilayer containing a single copy of an active channel in response to a voltage sweep (increment 5 mV/s) from 0 to +50 mV and from 0 to −80 mV. The cis compartment contained 2 M KCl, 10 mM MOPS–Tris pH 7.0, and the trans compartment contained 250 mM KCl, 10 mM MOPS–Tris pH 7.0. Dotted lines indicate the slope of two different channel conductance states. Download figure Download PowerPoint In bi-ionic measurements, we investigated the relative permeability of the OEP21 channel for 3-phosphoglycerate (3PGA), glucose-6-phosphate (G6P), triosephosphate (TP), HPO42− and others. All ions were able to permeate OEP21 (not shown). These measurements allowed calculation of the permeability ratios for those ions that carry a uniform charge under the experimental conditions (Table I) (Hille, 1968). Conversion of the measured reversal potentials into permeability ratios by the GHK constant field approach revealed higher permeability for the divalent anions SO42− and HPO42−. The apparent higher permeability of the divalent anions implies that they interact more strongly with the OEP21 channel pore than monovalent anions. Table 1. Selectivity of the OEP21 channel Ion Vrev (mV) Pion/PCl− K+a −21 0.33 ± 0.01 K+b −28 0.21 ± 0.01 NO2− −20 1.1 ± 0.1 NO3− −13 2.4 ± 0.4 SO42− 3 3.6 ± 1.5 HPO42− 0 3.9 ± 1.8 Malate −23 0.7 ± 0.3 a Reversal potential of the fully open channel. b Reversal potential of the most frequent subconductance state. Rectification and ion selectivity of the OEP21 channel are regulated by ATP/triosephosphate and other phosphates When 1 mM ATP was added to the trans compartment of the bilayer containing active OEP21 channels, we observed a drastic change in the reversal potential and the shape of the current–voltage relationship (Figure 5A, C and D). The addition of ATP resulted in a switch of positive to negative current at Vm = 0 mV (Figure 5A), i.e. rectification at positive voltages (see also Figure 4C) in the absence of ATP changed to rectification at negative voltages. Concomitantly, the ratio of slope conductance (a measure of the degree of rectification) of bilayers at positive (Vm > Vrev) and negative (Vm < Vrev) membrane potentials shows that 1–5 mM ATP when added to the trans compartment is sufficient at negative potentials to decrease anion currents from the trans to the cis compartment (Figure 5B). The permeability ratios in the absence or presence of 1 mM ATP changed from PCl−/PK+ ≅ 3:1 to PCl−/PK+ ≅ 1:4, respectively. We conclude that rectification of the OEP21 channel and its selectivity are regulated by ATP in a concentration-dependent manner from one side of the membrane only. GTP revealed the same changes as ATP on the rectification and reversal potential of reconstituted OEP21 (data not shown). When ATP was added to the cis compartment under the same ionic strength conditions (opposite gradient), neither a change in reversal potential nor a change in the profile of the current–voltage relationship of the OEP21 channel was observed (see Figure 5E and F). The site specificity of this effect was observed in >100 independent bilayer experiments. The ATP concentration dependence of this reversal potential change follows a typical saturation isotherm (Figure 6A and B). The data can be fitted by a two-site binding model: one high affinity binding site (Kb = 148 ± 38 μM ATP, ΔVmax = 23.6 mV) and a second lower affinity site of Kb = 18.5 ± 5 mM ATP, ΔVmax = 18.4 mV. Figure 5.Effec