Acetylcholine triggered enzymatic cascade reaction based on Fe7S8 nanoflakes catalysis for organophosphorus pesticides visual detection.

BACKGROUND: Organophosphorus pesticides (OPs) play important roles in the natural environment, agricultural fields, and biological prevention. The development of OPs detection has gradually become an effective strategy to avoid the dangers of pesticides abuse and solve the severe environmental and health problems in humans. Although conventional assays for OPs analysis such as the bulky instrument required analytical methods have been well-developed, it still remains the limitation of inconvenient, inefficient and lab-dependence analysis in real samples. Hence, there is an urgent demand to develop efficient detection methods for OPs analysis in real scenarios. RESULTS: Here, by virtue of the highly efficient catalytic performance in Fe7S8 nanoflakes (Fe7S8 NFs), we propose an OPs detection method that rationally integrated Fe7S8 NFs into the acetylcholine (ACh) triggered enzymatic cascade reaction (ATECR) for proceeding better detection performances. In this method, OPs serve as the enzyme inhibitors for inhibiting ATECR among ACh, acetylcholinesterase (AChE), and choline oxidase (CHO), then reduce the generation of H2O2 to suppress the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) that catalyzed by Fe7S8 NFs. Benefiting from the integration of Fe7S8 NFs and ATECR, it enables a sensitive detection for OPs (e.g. dimethoate). The proposed method has presented good linear ranges of OPs detection ranging from 0.1 to 10 µg mL-1. Compared to the other methods, the comparable limits of detection (LOD) of OPs are as low as 0.05 µg mL-1. SIGNIFICANCE: Furthermore, the proposed method has also achieved a favorable visual detection performance of revealing OPs analysis in real samples. The visual signals of OPs can be transformed into RGB values and gathered by using smartphones, indicating the great potential in simple, sensitive, instrument-free and on-site analysis of pesticide residues in environmental monitoring and biosecurity research.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Electrostatic Contributions to the Binding Free Energy of Nicotine to the Acetylcholine Binding Protein.

Biomolecular binding relies on specific attractive interactions between two partner molecules, including electrostatics, dispersion, hydrophobicity, and solvation. Assessing the contributions of electrostatic interactions to binding is key to the understanding of ligand binding mechanisms and the design of improved biomolecular binders. For example, nicotine is a well-known agonist of nicotinic acetylcholine receptors (nAChRs), but the molecular mechanisms for the differential action of nicotine on brain and muscle nAChRs remain elusive. In this work, we have chosen the acetylcholine binding protein (AChBP) in complex with nicotine as a model system to interrogate the electrostatic contributions to nicotine binding. Our absolute binding free energy simulations confirm that nicotine binds AChBP predominantly in its protonated (charged) form. By comparing energetic contributions from decomposed interactions for either neutral or charged nicotine, our calculations shed light on the nature of the binding of nicotine to the AChBP. The preferred binding of charged nicotine over neutral nicotine originates from its stronger electrostatic interactions with AChBP, a cation-π interaction to a tryptophan residue and a hydrogen bond between nicotine and the backbone carbonyl of the tryptophan, whereas the major force driving the binding process appears to be van der Waals interactions. The various nonelectrostatic terms can also indirectly modulate the electrostatic interactions through fine-tuning the binding pose of the ligand in the binding site, providing an explanation of why the binding specificity of nicotine to the brain versus muscle nAChRs is driven by electrostatic interaction, given that the immediate binding site residues, including the key tryptophan residue, are identical in the two receptors.

Delineating the activity of the potent nicotinic acetylcholine receptor agonists (+)-anatoxin-a and (-)-hosieine-A.

The affinity and thermodynamic parameters for the interactions of two naturally occurring neurotoxins, (+)-anatoxin-a and (-)-hosieine-A, with acetylcholine-binding protein were investigated using a fluorescence-quenching assay and isothermal titration calorimetry. The crystal structures of their complexes with acetylcholine-binding protein from Aplysia californica (AcAChBP) were determined and reveal details of molecular recognition in the orthosteric binding site. Comparisons treating AcAChBP as a surrogate for human α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the molecular features involved in ligand recognition and affinity for the protein targets are conserved. The ligands exploit interactions with similar residues as the archetypal nAChR agonist nicotine, but with greater affinity. (-)-Hosieine-A in particular has a high affinity for AcAChBP driven by a favorable entropic contribution to binding. The ligand affinities help to rationalize the potent biological activity of these alkaloids. The structural data, together with comparisons with related molecules, suggest that there may be opportunities to extend the hosieine-A scaffold to incorporate new interactions with the complementary side of the orthosteric binding site. Such a strategy may guide the design of new entities to target human α4ß2 nAChR that may have therapeutic benefit.

Molecular Interactions between an Enzyme and Its Inhibitor for Selective Detection of Limonene.

Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonene─the major odorant of citrus and an AChE inhibitor─using molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.

Interactions between 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues and acetylcholine-binding protein inform on potent antagonist activity against nicotinic receptors.

Low-nanomolar binding constants were recorded for a series of six 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues with acetylcholine-binding protein (AChBP). The crystal structures of three complexes with AChBP reveal details of molecular recognition in the orthosteric binding site and imply how the other three ligands bind. Comparisons exploiting AChBP as a surrogate for α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the key interactions are conserved. The ligands interact with the same residues as the archetypal nAChR agonist nicotine yet display greater affinity, thereby rationalizing their in vivo activity as potent antagonists of nicotine-induced antinociception. An oxyanion-binding site is formed on the periphery of the AChBP orthosteric site by Lys42, Asp94, Glu170 and Glu210. These residues are highly conserved in the human α4, ß2 and α7 nAChR sequences. However, specific sequence differences are discussed that could contribute to nAChR subtype selectivity and in addition may represent a point of allosteric modulation. The ability to engage with this peripheral site may explain, in part, the function of a subset of ligands to act as agonists of α7 nAChR.

A sensitive approach for screening acetylcholinesterase inhibition of water samples using ultra-performance liquid chromatography-tandem mass spectrometry.

A sensitive assay was developed to evaluate inhibitory effects of aqueous solution on acetylcholinesterase (AChE) activity via measuring hydrolysis rates of acetylcholine (ACh) based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Upon having identified precursor ions and product ions of the ACh and its hydrolysis products choline (Ch), the separation chromatogram for these two analytes has been established using a 50 mm reverse-phase BEH Shield RP18 column. The total chromatographic separation time is 7 min; limits of detection (LODs) for ACh and Ch are 0.14 µg L-1 and 0.12 µg L-1, respectively. A simple method for inactivation of AChE and optimization of operational parameters were then sequentially performed. It was found that adjusting solution pH to 2.5 not only can terminate the enzymatic reaction but also solve band shifting and broadening caused by aqueous matrices in chromatographic separation during UPLC-MS/MS detection. Under conditions of 0.00075 U mL-1 AChE, initial concentration of ACh at 100 µg L-1 and 20 min observation time, IC50 values of the proposed assay for chlorpyrifos-oxon, diazoxon, malaoxon, methidathion oxon, omethoate and paraoxon were 3.5 nM, 16.8 nM, 2.4 nM, 6.8 nM, 270 nM and 36.9 nM, respectively. They are 4.5-51.9 times smaller than those reported in a LC-MS based method, and >120 times lower than those obtained by the traditional Ellman method. The results suggested that, the proposed assay significantly increases the sensitivity of commercial AChE. In addition, inhibition efficiencies of three surface waters, a groundwater and four commercial brands of bottled drinking water samples on AChE activity were firstly measured using this UPLC-MS/MS based method. These water samples were proved to have different inhibitory effects on AChE activity, and the inhibition efficiencies dependent on concentrations of dissolved organic carbon (DOC) but are independent of UV absorbance at 254 nm (UV254) values. These results indicate that the proposed method has advantages of high sensitivity over all other conventional methods. It may become a promising AChE inhibition assay for assessing toxicity of aqueous solution containing neurotoxicity contaminants such as organophosphorus pesticides (OPPs) at low levels, or used to evaluate potential inhibition effects of natural waters on AChE activity.

Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity.

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site ("PVsite") were deduced.In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.

From Crystal Structures of RgIA4 in Complex with Ac-AChBP to Molecular Determinants of Its High Potency of α9α10 nAChR.

α9-containing nicotinic acetylcholine receptors (nAChRs) have been shown to play critical roles in neuropathic pain. The α-conotoxin (α-CTx) RgIA and its analog RgIA4 were identified as the most selective inhibitor of α9α10 nAChR. However, the mechanism of their selectivity toward α9α10 nAChR remains elusive. Here, we reported the co-crystal structure of RgIA and RgIA4 in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at resolution of 2.6 Å, respectively. Based on the structure of the complexes, together with molecular dynamic simulation (MD-simulation), we suggested the key residues of α9α10 nAChR in determining its high affinity for RgIA/RgIA4. This is the first time the complex between pain-related conotoxins and Ac-AChBP was reported and the complementary side of α9 subunit in binding of the antagonists shown. These results provide realistic template for the design of new therapeutic in neuropathic pain.

Exploring the Binding Pattern of Geraniol with Acetylcholinesterase through In Silico Docking, Molecular Dynamics Simulation, and In Vitro Enzyme Inhibition Kinetics Studies.

Acetylcholinesterase (AChE) inhibition is a key element in enhancing cholinergic transmission and subsequently relieving major symptoms of several neurological and neuromuscular disorders. Here, the inhibitory potential of geraniol and its mechanism of inhibition against AChE were elucidated in vitro and validated via an in silico study. Our in vitro enzyme inhibition kinetics results show that at increasing concentrations of geraniol and substrate, Vmax did not change significantly, but Km increased, which indicates that geraniol is a competitive inhibitor against AChE with an IC50 value 98.06 ± 3.92 µM. All the parameters of the ADME study revealed that geraniol is an acceptable drug candidate. A docking study showed that the binding energy of geraniol (-5.6 kcal mol-1) was lower than that of acetylcholine (-4.1 kcal mol-1) with AChE, which exhibited around a 12.58-fold higher binding affinity of geraniol. Furthermore, molecular dynamics simulation revealed that the RMSD of AChE alone or in complex with geraniol fluctuated within acceptable limits throughout the simulation. The mean RMSF value of the complex ensures that the overall conformation of the protein remains conserved. The average values of Rg, MolSA, SASA, and PSA of the complex were 3.16 Å, 204.78, 9.13, and 51.58 Å2, respectively. We found that the total SSE of AChE in the complex was 38.84% (α-helix: 26.57% and ß-sheets: 12.27%) and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.

Improvement of the Solubilization and Extraction of Curcumin in an Edible Ternary Solvent Mixture.

A water-free, ternary solvent mixture consisting of a natural deep eutectic solvent (NADES), ethanol, and triacetin was investigated concerning its ability to dissolve and extract curcumin from Curcuma longa L. To this purpose, 11 NADES based on choline chloride, acetylcholine, and proline were screened using UV-vis measurements. A ternary phase diagram with a particularly promising NADES, based on choline chloride and levulinic acid was recorded and the solubility domains of the monophasic region were examined and correlated with the system's structuring via light scattering experiments. At the optimum composition, close to the critical point, the solubility of curcumin could be enhanced by a factor of >1.5 with respect to acetone. In extraction experiments, conducted at the points of highest solubility and evaluated via HPLC, a total yield of ~84% curcuminoids per rhizome could be reached. Through multiple extraction cycles, reusing the extraction solvent, an enrichment of curcuminoids could be achieved while altering the solution. When counteracting the solvent change, even higher concentrated extracts can be obtained.

The Effects of Structural Alterations in the Polyamine and Amino Acid Moieties of Philanthotoxins on Nicotinic Acetylcholine Receptor Inhibition in the Locust, Schistocerca gregaria.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 µM at -75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 µM at -75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 µM at -75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at -75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 µM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Reducing Passive Drug Diffusion from Electrophoretic Drug Delivery Devices through Co-Ion Engineering.

Implantable electrophoretic drug delivery devices have shown promise for applications ranging from treating pathologies such as epilepsy and cancer to regulating plant physiology. Upon applying a voltage, the devices electrophoretically transport charged drug molecules across an ion-conducting membrane out to the local implanted area. This solvent-flow-free "dry" delivery enables controlled drug release with minimal pressure increase at the outlet. However, a major challenge these devices face is limiting drug leakage in their idle state. Here, a method of reducing passive drug leakage through the choice of the drug co-ion is presented. By switching acetylcholine's associated co-ion from chloride to carboxylate co-ions as well as sulfopropyl acrylate-based polyanions, steady-state drug leakage rate is reduced up to sevenfold with minimal effect on the active drug delivery rate. Numerical simulations further illustrate the potential of this method and offer guidance for new material systems to suppress passive drug leakage in electrophoretic drug delivery devices.

Concatemers to re-investigate the role of α5 in α4ß2 nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, ß2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4ß2 and α4ß2α5 receptors and characterized their properties by two-electrodes voltage-clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and ß2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.

Polymeric Membrane Electrodes Using Calix[4]pyrrole Bis/Tetra-Phosphonate Cavitands as Ionophores for Potentiometric Acetylcholine Sensing with High Selectivity.

A handful of bis/tetra-phosphonate calix[4]pyrroles with recognition sites embedding in hydrophobic cavitands were evaluated for the first time as ionophores for polymeric membrane Ach+-selective electrodes. Highly selective potentiometric Ach+ could be achieved over its analogues, especially for Ch+, which differs only by an acetate tail from Ach+. The superior performance of the proposed ISEs might be ascribed to a dual-site binding mode, in which the trimethylammonium head and acetate tail were accommodated by the phosphonate group-bridged aryl walls and the bowl-shaped aromatic cavity, through cation-π/charge-dipole interaction and the convergent four N-H···O hydrogen bonds, respectively. To gain more insight into the performance of the proposed ISEs, the cation-ionophore complex constants in the membrane phase were determined, and the binding affinity trend correlates well with the selectivity pattern. These results suggest that conformational preorganization of the ionophores and complementary weak interactions do change the selectivity of the ionophores. Studies on the influence of the sample solution pH demonstrated that the developed ISEs can be employed in a wide pH range of 4.0-9.6 with a fast response (<60 s), good reversibility, and long lifetime. Optimizing the membrane components, such as ionophores, lipophilic additives, and plasticizers, yielded ISEs, showing Nernstian responses to Ach+ with improved linear ranges and detection limits (a slope of -59.5 mV/dec in the linear range of 1 × 10-6-1 × 10-2 M with a detection limit of 3 × 10-7 M), which led to the success of the determination of Ach+ in spiked urine and milk samples.

Kinetics of Chemical Processes in the Human Brain. The Cholinergic Synapse-Mechanisms of Functioning and Control Methods.

In the framework of the kinetic model, the functioning of the cholinergic synapse is considered. The results of mathematical modeling of changes in the level of acetylcholine, induced pH impulse, the influence of the frequency of impulse transmission and inhibition of acetylcholinesterase are presented. Physicochemical explanation for a number of important physiological phenomena, such as neuromuscular paralysis, the molecular mechanism of neurological memory, and actions of nerve poisons and toxins, is given.

Quaternary-Ammonium-Modulated Surface-Enhanced Raman Spectroscopy Effect: Discovery, Mechanism, and Application for Highly Sensitive In Vitro Sensing of Acetylcholine.

Quaternary ammonium (QA) plays multiple roles in biological functions, whose dysregulation may result in multiple diseases. However, how to efficiently detect QA-based materials such as acetylcholine (ACh) still remains a great challenge, especially in complex biological environments. Here, a new effect [called quaternary-ammonium-modulated surface-enhanced Raman spectroscopy (QAM-SERS) effect] is discovered, showing that the existence of QA will modulate the intensity of SERS signals in a concentration-dependent manner. When the QAM-SERS effect is used, a new method is easily developed for in vitro detection of ACh with an extremely high sensitivity and an ultrawide dynamic range. Particularly, the linear dynamic range can be freely tuned to adapt for various physiological samples. As a proof-of-concept experiment, the time-dependent secretion of ACh from PC12 cells was successfully monitored using the QAM-SERS method, which were under either the stimulation of potassium ions or the incubation of drugs. The discovery of the QAM-SERS effect provides an easy and universal strategy for detecting ACh as well as other QA-contained molecules, which can also inspire new insights into the roles that QA could play in biology and chemistry.

Acetylcholine and Acetylcholine Receptors: Textbook Knowledge and New Data.

It was a pleasure to receive a proposal to organize and be a guest editor of a Special Issue ofBiomolecules. This is the field in which I am working and personally know some of the leadingscientists. My narrow field is the research on the peptide and protein neurotoxins from animalvenoms and their application as sophisticated tools for analysis of nicotinic acetylcholine receptors(nAChRs) [...].

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.