A bupropion modulatory site in the Gloeobacter violaceus ligand-gated ion channel.

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels, such as nicotinic acetylcholine and serotonin type 3A receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pentameric ligand-gated ion channels, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using the GLIC as a model, we used molecular docking to predict binding sites for bupropion. Bupropion was found to bind to several sites within the transmembrane domain, with the predominant site being localized to the interface between transmembrane segments M1 and M3 of two adjacent subunits. Residues W213, T214, and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within a 4 Å distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the half-maximal inhibitory concentration to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in the GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in γ-aminobutyric acid type A receptors. Our data thus suggest that the T214 that lines a previously identified lipophilic binding pocket in GLIC and γ-aminobutyric acid type A receptors is also a modulatory site for bupropion interaction with the GLIC.

Identification of a binding site for bupropion in Gloeobacter violaceus ligand-gated ion channel.

Bupropion is an atypical antidepressant and smoking cessation drug which causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. However, the binding sites and binding mechanisms of bupropion are still elusive. To further understand the inhibition of pLGICs by bupropion, in this work, using a prokaryotic homologue of pLGICs as a model, we examined the inhibitory potency of bupropion in Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated ion channel. Bupropion inhibited proton-induced currents in GLIC with an inhibitory potency of 14.9 ± 2.0 µM, comparable to clinically attainable concentrations previously shown to also modulate eukaryotic pLGICs. Using single amino acid substitutions in GLIC and two-electrode voltage-clamp recordings, we further determined a binding site for bupropion in the lower third of the first transmembrane segment M1 at residue T214. The sidechain of M1 T214 together with additional residues of M1 and also of M3 of the adjacent subunit have previously been shown to contribute to binding of other lipophilic molecules like allopregnanolone and pregnanolone.

Binding motif for RIC-3 chaperon protein in serotonin type 3A receptors.

Serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors belong to the family of pentameric ligand-gated ion channels (pLGICs) that are therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities of pLGICs' extracellular and transmembrane domains, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A subunit intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In the present study, synthetic L1-MX-based peptides and Ala-scanning identify positions W347, R349, and L353 as critical for binding to RIC-3. Complementary studies using full-length 5-HT3A subunits confirm that the identified Ala substitutions reduce the RIC-3-mediated modulation of functional surface expression. Additionally, we find and characterize a duplication of the binding motif, DWLR…VLDR, present in both the MX-helix and the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary, we identify the binding motif for RIC-3 in 5-HT3A subunits at two locations in the ICD, one in the MX-helix and one at the MAM4-helix transition.

Cell-Free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs.

Proton coupled folate transporter (PCFT) is an integral membrane protein with 12 transmembrane segments localized to the plasma membrane. PCFT is the main route by which folate, vitamin B9, from dietary sources enters mammalian cells in the small intestine. Loss-of-function mutations in this membrane transport protein cause hereditary folate malabsorption, and upregulation of PCFT has been reported in cancer cells. Currently, a complete translocation mechanism of folate via PCFT is still missing. To reveal this mechanism via studies of structural architecture and structure-function relationships, soluble and stable PCFT in a phospholipid bilayer environment is needed. We therefore develop an approach to screen lipid environments in which PCFT is most soluble. Traditional in vitro expression and reconstitution into lipid bilayers of integral membrane proteins requires separate steps, which are costly and time-consuming. In this chapter, we describe a protocol for in vitro translation of PCFT into preformed lipid nanodiscs using a cell-free expression system, which helps to accelerate and reduce the cost of the sample preparation.

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Mobility of Lower MA-Helices for Ion Conduction through Lateral Portals in 5-HT3A Receptors.

The intracellular domain of the serotonin type 3A receptor, a pentameric ligand-gated ion channel, is crucial for regulating conductance. Ion permeation through the extracellular vestibule and the transmembrane channel is well understood, whereas the specific ion conduction pathway through the intracellular domain is less clear. The intracellular domain starts with a short loop after the third transmembrane segment, followed by a short α-helical segment, a large unstructured loop, and finally, the membrane-associated MA-helix that continues into the last transmembrane segment. The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a "closed vestibule," with their lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. The question remains whether the lateral portals or cytosolic constriction conduct ions upon channel opening. In our study, we used disulfide bond formation between pairs of engineered cysteines to probe the proximity and mobility of segments of the MA-helices most distal to the membrane bilayer. Our results indicate that the proximity and orientation for cysteine pairs at I409C/R410C, in close proximity to the lateral windows, and L402C/L403C, at the cytosolic ends of the MA-helices, are conducive for disulfide bond formation. Although conformational changes associated with gating promote cross-linking for I409C/R410C, which in turn decreases channel currents, cross-linking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely.

Bupropion Inhibits Serotonin Type 3AB Heteromeric Channels at Clinically Relevant Concentrations.

Bupropion, a Food and Drug Administration-approved antidepressant and smoking cessation aid, blocks dopamine and norepinephrine reuptake transporters and noncompetitively inhibits nicotinic acetylcholine and serotonin (5-HT) type 3A receptors (5-HT3ARs). 5-HT3 receptors are pentameric ligand-gated ion channels that regulate synaptic activity in the central and peripheral nervous system, presynaptically and postsynaptically. In the present study, we examined and compared the effect of bupropion and its active metabolite hydroxybupropion on mouse homomeric 5-HT3A and heteromeric 5-HT3AB receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp experiments. Coapplication of bupropion or hydroxybupropion with 5-HT dose dependently inhibited 5-HT-induced currents in heteromeric 5-HT type 3AB receptors (5-HT3ABRs) (IC50 = 840 and 526 µM, respectively). The corresponding IC50s for bupropion and hydroxybupropion for homomeric 5-HT3ARs were 10- and 5-fold lower, respectively (87 and 113 µM). The inhibition of 5-HT3ARs and 5-HT3ABRs was non-use dependent and voltage independent, suggesting bupropion is not an open channel blocker. The inhibition by bupropion was reversible and time-dependent. Of note, preincubation with a low concentration of bupropion that mimics therapeutic drug conditions inhibits 5-HT-induced currents in 5-HT3A and 5-HT3AB receptors considerably. In summary, we demonstrate that bupropion inhibits heteromeric 5-HT3ABRs as well as homomeric 5-HT3ARs. This inhibition occurs at clinically relevant concentrations and may contribute to bupropion's clinical effects. SIGNIFICANCE STATEMENT: Clinical studies indicate that antagonizing serotonin (5-HT) type 3AB (5-HT3AB) receptors in brain areas involved in mood regulation is successful in treating mood and anxiety disorders. Previously, bupropion was shown to be an antagonist at homopentameric 5-HT type 3A receptors. The present work provides novel insights into the pharmacological effects that bupropion exerts on heteromeric 5-HT3AB receptors, in particular when constantly present at low, clinically attainable concentrations. The results advance the knowledge on the clinical effects of bupropion as an antidepressant.

Delineating the Site of Interaction of the 5-HT3A Receptor with the Chaperone Protein RIC-3.

The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains: extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Previously, we have demonstrated that the 5-HT3A-ICD is required for the interaction between 5-HT3A and the chaperone protein resistance to inhibitors of choline esterase (RIC-3). Additionally, we have shown that 5-HT3A-ICD fused to maltose-binding protein (MBP) directly interacts with RIC-3, without the involvement of other protein(s). To elucidate the molecular determinants of this interaction, we developed different MBP-fused 5-HT3A-ICD constructs by deleting large segments of its amino acid sequence. We expressed seven engineered ICDs in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction between MBP-5HT3A-ICD constructs and RIC-3 was investigated. In summary, we identify a 24-amino-acid-long segment of the 5-HT3A-ICD as a molecular determinant for the interaction between the 5-HT3A-ICD and RIC-3.

Triple arginines as molecular determinants for pentameric assembly of the intracellular domain of 5-HT3A receptors.

Serotonin type 3 receptors (5-HT3Rs) are cation-conducting pentameric ligand-gated ion channels and members of the Cys-loop superfamily in eukaryotes. 5-HT3Rs are found in the peripheral and central nervous system, and they are targets for drugs used to treat anxiety, drug dependence, and schizophrenia, as well as chemotherapy-induced and postoperative nausea and emesis. Decades of research of Cys-loop receptors have identified motifs in both the extracellular and transmembrane domains that mediate pentameric assembly. Those efforts have largely ignored the most diverse domain of these channels, the intracellular domain (ICD). Here we identify molecular determinants within the ICD of serotonin type 3A (5-HT3A) subunits for pentameric assembly by first identifying the segments contributing to pentamerization using deletion constructs of, and finally by making defined amino acid substitutions within, an isolated soluble ICD. Our work provides direct experimental evidence for the contribution of three intracellular arginines, previously implicated in governing the low conductance of 5-HT3ARs, in structural features such as pentameric assembly.

A modified clear-native polyacrylamide gel electrophoresis technique to investigate the oligomeric state of MBP-5-HT3A-intracellular domain chimeras.

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT3A) receptors.

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1).

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors.

The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT3ARs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT3ARs were heterologously expressed in HEK-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT3ARs reversibly and dose-dependently with inhibitory potencies of 87 µM and 112 µM, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect.

Experimentally optimized threading structures of the proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X-ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol-3-phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head-groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward-open. Several positions lining the substrate cavity have been identified. Motif A, a helix-turn-helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward-open state. A charge-relay system between three charged residues as well as apposing glycines in two α-helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward-open state of a putative proton-driven transporter (YajR).

Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors.

In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5-HT3A ) that the interaction with RIC-3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC-3 attenuated serotonin-induced currents in 5-HT3A wild-type channels, but not in functional 5-HT3A glvM3M4 channels that have the 115-amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand-gated ion channel (GLIC) to contain the 5-HT3A -ICD confers sensitivity to RIC-3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC-3 protein expression in X. laevis oocytes. We purified RIC-3 to homogeneity after expression in Echericia coli. By using heterologously over-expressed and purified RIC-3 and the chimera consisting of the 5-HT3A -ICD and the extracellular and transmembrane domains of GLIC in pull-down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5-HT3 A R that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3 A R-ICD and RIC-3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC-5-HT3A -ICD chimera interacts with RIC-3. We hypothesized that pentameric ligand-gated ion channels (pLGICs) associate directly with the chaperone protein RIC-3 (resistance to inhibitors of cholinesterase type 3), and that the interaction does not require other protein factors. We found that the two proteins indeed interact directly, that the pLGIC intracellular domain is required for the effect, and that pLGICs in their pentameric form associate with RIC-3. These results provide the basis for future studies aimed at investigating which motifs provide the interaction surfaces, and at delineating the mechanism(s) of RIC-3 modulation of functional pLGIC surface expression.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity--14% or less--between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology/threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.

The monomeric state of the proton-coupled folate transporter represents the functional unit in the plasma membrane.

Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [(3)H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin-solubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT(3A) receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.