A catalytically silent FAAH-1 variant drives anandamide transport in neurons

Abstract

The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, that remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), that lacked amidase activity but bound anandamide with low micromolar affinity and facilitated its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, blocked these effects. We also identified a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, that prevented anandamide internalization in vitro, interrupted anandamide deactivation in vivo and exerted profound analgesic effects in rodent models of nociceptive and inflammatory pain, which were mediated by CB(1) cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.

Fig. 1

Structural properties of FLAT. (a) Predicted amino acid sequences of FLAT and FAAH-1; residues comprising the catalytic triad of FAAH-1 (Lys¹⁴², Ser²¹⁷ and nucleophile Ser²⁴¹) are highlighted. (b) Model of rat FLAT (left) based on the structure of FAAH-ΔTM (right), a FAAH-1 mutant lacking the α1 helix which is redrawn in green for illustration purposes. Most of the α1 helix and the entire α2 helix (orange) of FAAH-1 are absent in FLAT. Both FAAH-1 and FLAT contain a membrane-binding domain (blue, FLAT residues 343–367) and an 'α2-interacting loop' (red, FLAT residues 187–210), which may interact with the α2 helix and help shield the enzyme's catalytic pocket from water. The membrane model was generated using Molecular Dynamics simulations of a 1,2-dioleoyl-sn-glycerol-3-phosphorylcholine bilayer.

Fig. 2

FLAT binds to anandamide and facilitates its transport into cells. (a) Specific binding of [³H]-anandamide to rat FLAT-glutathione-S-transferase (GST) (closed squares) or GST alone (open squares). The inset shows a Scatchard transformation (bound - [bound/free], in nmol) of binding data. (b) AM404 (closed squares) and OMDM-1 (closed squares) antagonize [³H]-anandamide binding to FLAT-GST, whereas URB597 (open circles) has no effect. (c) Cytosolic fractions of FLAT-expressing Hek293 cells contain detectable amounts of FLAT (arrow); a corresponding fraction from FAAH-1-expressing cells is shown for comparison. The lower band is β-actin. (d) [³H]- Anandamide accumulation in control cells (vector-transfected, open bar) or FLAT-expressing Hek293 cells (closed bars) incubated with vehicle (0.01%dimethylsulfoxide), AM404 or non-radioactive anandamide (concentrations in μM). (e) The anandamide transport inhibitors OMDM-1, UCM-707 and VDM-11 suppress [³H]-anandamide accumulation in FLAT-expressing Hek293 cells. This process is not affected by URB597 or mutation of catalytic Ser²⁴² (shaded bar). (f) Accumulation of labeled lipids in control (open bars) or FLAT-expressing Hek293 cells (closed bars). Abbreviations: [³H]-anandamide, AEA; [³H]-oleoylethanolamide, OEA; [³H]-palmitoylethanolamide, PEA; [³H]-arachidonic acid, AA; [³H]-2-arachidonoyl-sn-glycerol, 2-AG. Results are expressed as mean±SEM of 3-7 experiments. ***P<0.01, versus vector-transfected cells, Student's t test; ^#P<0.05; ^##, P<0.01; ^###, P<0.001 versus vehicle, one-way ANOVA followed by Dunnett's test.

Fig. 3

ARN272 is a competitive FLAT inhibitor. (a) Effects of ARN272 on [³H]-anandamide binding to FLAT-GST. The inset shows the chemical structure of ARN272. (b) Effects of ARN272 (concentrations in μM) on [³H]-anandamide accumulation in FLAT-expressing Hek-293 cells (closed bars); the open bar represents vector-transfected cells. (c) Effects of vehicle (open bar), ARN272 and AM404 (closed bars) on [³H]-anandamide accumulation in rat cortical neurons in cultures. (d) Effects of ARN272 and URB597 on FAAH activity in rat brain membranes. (e, f) Effects of ARN272 (1 mg-kg⁻¹, i.p.) on plasma levels of (e) anandamide or (f) OEA, PEA and 2-AG in mice. Results are expressed as mean±SEM of 3–7 experiments. ***, P<0.001 versus vector-transfected cells, Student's t test; ^#, P<0.05 and ^##, P<0.01 versus vehicle; one-way ANOVA followed by Dunnett's test.

Fig. 4

ARN272 produces CB₁-dependent antinociception in mice. Intraplantar injection of formalin (5%, 20 μL) elicited two temporally distinct phases of nocifensive behavior in mice: phase I (0–5 min, open bars) and phase II (5–45 min, closed bars). (a) ARN272 (doses in mg-kg⁻¹, i.p.) decreased nocifensive behavior in both phases. (b) The CB₁ antagonist AM251 (1 mg-kg⁻¹, i.p.) abolished the antinociceptive effects of ARN272, whereas the CB₂ antagonist AM630 and the TRPV1 antagonist AMG9810 did not. (c–f) Intraplantar injection of carrageenan (car) elicited a local inflammatory response in mice. ARN272 (mg-kg⁻¹, i.p.) decreased (c) thermal hyperalgesia (withdrawal latency, in seconds), and (e) edema (volume, in ml). The CB₁ antagonist AM251 (1 mg-kg⁻¹, i.p.) suppressed the effects of ARN272 on (d) thermal hyperalgesia and (f) edema. Results are expressed as the mean±SEM of 6 mice per group. *, P<0.05; **, P<0.01; ***, P<0.001 versus vehicle-injected controls; two-way ANOVA followed by Bonferroni's test.