Axonal targeting of the 5-HT1B serotonin receptor relies on structure-specific constitutive activation.

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.

Modulation of 5-HT3 receptor desensitization by the light chain of microtubule-associated protein 1B expressed in HEK 293 cells.

Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins has been the topic of recent research. Here, we report that the light chain (LC1) of microtubule-associated protein 1B (MAP1B) specifically interacted with the 5-HT(3A) receptor, a predominant serotonin-gated ion channel in the brain. LC1 and 5-HT(3A) receptors were colocalized in central neurons and in HEK 293 cells expressing 5-HT(3A) receptors. LC1 reduced the steady-state density of 5-HT(3A) receptors at the membrane surface of HEK 293 cells and significantly accelerated receptor desensitization time constants from 3.8 +/- 0.3 s to 0.8 +/- 0.1 s. However, LC1 did not significantly alter agonist binding affinity and single-channel conductance of 5-HT(3A) receptors. On the other hand, application of specific LC1 antisense oligonucleotides and nocodazole, a microtubule disruptor, significantly prolonged the desensitization time of the recombinant and native neuronal 5-HT(3) receptors by 3- to 6-fold. This kinetic change induced by nocodazole was completely rescued by addition of LC1 but not GABA(A) receptor-associated protein (GABARAP), suggesting that LC1 can specifically interact with 5-HT(3A) receptors. These observations suggest that the LC1-5-HT(3A) receptor interaction contributes to a mechanism that regulates receptor desensitization kinetics. Such dynamic regulation may play a role in reshaping the efficacy of 5-HT(3) receptor-mediated synaptic transmission.

Immunolabelling of the 5-HT 3B receptor subunit in the central and peripheral nervous systems in rodents.

The 5-HT(3) receptor is a member of the superfamily of neurotransmitter-gated ion channels involved in fast synaptic signalling and in modulation of neurotransmitter release. As for many other channel receptors, the electrophysiological properties and the functions of the 5-HT(3) receptor are determined by subunit composition of the pentameric channel. Because in situ hybridization did not allow the detection of mRNA encoding the 5-HT(3B) subunit in the rodent central nervous system, or in nearly half of the neurons expressing the 5-HT(3A) subunit in peripheral ganglia, it has been suggested that subunit composition could define at least two 5-HT(3) receptor-expressing neuronal populations. In order to challenge this hypothesis, we have developed polyclonal antibodies directed against a portion of the second intracytoplasmic loop of the mouse 5-HT(3B) subunit. Immunohistochemical analysis in the mouse and the rat revealed that immunolabelling was most prominent in peripheral ganglia, particularly in trigeminal ganglia (TG). In rats, transection or ligature of the infraorbital nerve resulted in a pronounced accumulation of immunoreactive material at the proximal side of the lesioned nerve, and an up-regulation of both subunits in 5-HT(3) receptor-expressing TG neurons. Surprisingly, nearly 100% of neurons expressing 5-HT(3A) subunits were also labelled by anti-5-HT(3B) antibodies. We also detected 5-HT(3B) immunoreactivity in the rat hippocampal CA1 layer and in scattered cortical neurons, indicating that detection of 5-HT(3) subunit mRNA by in situ hybridization might not provide really complete mapping of heteromeric 5-HT(3A/B) vs. homomeric 5-HT(3A) receptors in the peripheral and central nervous systems in rodents.

A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface.

Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels.

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.

Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels.

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.

Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels.

Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the functional interaction between native P2X2 and 5-HT3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.

Native and cloned 5-HT(3A)(S) receptors are anchored to F-actin in clonal cells and neurons.

Using selective antibodies to visualize the short isoform of the 5-HT(3A) receptor, we report here that both native and cloned 5-HT(3A)(S) receptors formed clusters associated with F-actin in all cell types studied. NG 108-15 cells expressing native 5-HT(3A)(S) receptors, COS-7 cells transiently expressing 5-HT(3A)(S) subunits, and CHO cells stably transfected with a plasmid encoding the 5-HT(3A)(S) sequence all exhibited similar surface receptor topology with 5-HT(3A)(S) receptor cluster accumulation in F-actin-rich lamellipodia and microspikes. Colocalization and coclustering of 5-HT(3A)(S) subunits and F-actin were also observed in transfected hippocampal neurons. Treatment of the neurons with latrunculin-A, a compound altering F-actin polymerization, demonstrated that 5-HT(3A)(S) receptor cluster size and topology were dependent on F-actin integrity. These results suggest that the anchoring of 5-HT(3A)(S) receptor clusters to the cytoskeletal network probably plays a key role in the physiological regulation of the receptor topology and dynamics, as is the case for other members of the 4-TMD ion channel receptor family.