Diego Rayes

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.

Number and Locations of Agonist Binding Sites Required to Activate Homomeric Cys-Loop Receptors

Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

Relationship between α7 nAChR and apoptosis in human lymphocytes

Abstract The presence of nicotinic receptors (nAChRs) in blood cells has been demonstrated. However, little is known about their functional roles. We have detected mRNA of α7 nAChR in peripheral human lymphocytes and determined that its expression is highly variable among individuals and within the same individual at different times. Upregulation of α7 is systematically observed after incubation of lymphocytes with nicotine or α-bungarotoxin. In addition, the incubation with these drugs decreases the percentage of apoptotic cells induced by the exposure to cortisol. Our results suggest that α7 nAChRs are involved in the modulation of cortisol-induced apoptosis.

MOLECULAR DETERMINANTS OF PYRANTEL SELECTIVITY IN NICOTINIC RECEPTORS

Nicotinic receptors (acetylcholine receptors, AChRs) play key roles in synaptic transmission throughout the nervous system. AChRs mediate neuromuscular transmission in nematodes, and they are targets for antiparasitic drugs. The anthelmintic agents levamisole and pyrantel, which are potent agonists of nematode muscle AChRs, are partial agonists of mammalian muscle AChRs. To further explore the structural basis of the differential activation of AChR subtypes by anthelmintics, we studied the activation of α7 AChRs using the high-conductance form of the α7-5-hydroxytryptamine-3A receptor, which is a good model for pharmacological studies involving the extracellular region of α7. Macroscopic and single-channel current recordings show that levamisole is a weak agonist of α7. It is interesting that pyrantel is a more potent agonist of α7 than acetylcholine (ACh). To identify determinants of this differential activation, we replaced residues of the complementary face of the binding site by the homologous residues in the muscle ϵ subunit and evaluated changes in activation. The mutation Q57G does not affect the activation by either ACh or levamisole. However, it increases EC50 values and decreases the maximal response to pyrantel. Kinetic analysis shows that gating of the mutant channel activated by pyrantel is profoundly impaired. The decreased sensitivity of α7-Q57G to pyrantel agrees with its weak action at muscle AChRs, indicating that when glycine occupies position 57, as in the mammalian muscle AChR, pyrantel behaves as a partial agonist. Thus, position 57 located at the complementary face of the binding site plays a key role in the selective activation of AChRs by pyrantel.

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

A Cys-loop Mutation in the Caenorhabditis elegans Nicotinic Receptor Subunit UNC-63 Impairs but Does Not Abolish Channel Function

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.

A Change in the Ion Selectivity of Ligand-Gated Ion Channels Provides a Mechanism to Switch Behavior.

Behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections. However, it is not known whether network formation and stability is constrained by the sign of synaptic connections between neurons within the network. Here we show that switching the sign of a synapse within a neural circuit can reverse the behavioral output. The inhibitory tyramine-gated chloride channel, LGC-55, induces head relaxation and inhibits forward locomotion during the Caenorhabditis elegans escape response. We switched the ion selectivity of an inhibitory LGC-55 anion channel to an excitatory LGC-55 cation channel. The engineered cation channel is properly trafficked in the native neural circuit and results in behavioral responses that are opposite to those produced by activation of the LGC-55 anion channel. Our findings indicate that switches in ion selectivity of ligand-gated ion channels (LGICs) do not affect network connectivity or stability and may provide an evolutionary and a synthetic mechanism to change behavior.

Imprinting: When Early Life Memories Make Food Smell Bad

A recent study has found that pathogen exposure early in the life of the nematode Caenorhabditis elegans leads to a long-lasting aversion that requires distinct sets of neurons for the formation and retrieval of the imprinted memory.

Co-option of neurotransmitter signaling for inter-organismal communication in C. elegans

Biogenic amine neurotransmitters play a central role in metazoan nervous systems, and both their chemical structures and cognate receptors are evolutionarily highly conserved. In the nematode C. elegans, four classical neurotransmitters - serotonin, dopamine, octopamine, and tyramine - have been detected and appear to serve signaling functions related to those in insects or vertebrates (1). Interestingly, one of the small molecule pheromones released by C. elegans incorporates the monoamine octopamine. Octopamine succinylated ascaroside #9 (osas#9) is biochemically derived by connecting the neurotransmitter octopamine to an ascaroside – a universal building block of pheromones in C. elegans (2, 3). Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2, a gene coding for a G protein-coupled receptor (GPCR), expression in the nociceptive neuron ASH is both necessary and sufficient to induce avoidance of osas#9. In contrast, expression of tyra-2 in AWA, a neuron pair primarily involved in attraction, reverses the behavioral response to osas#9. These results show that TYRA-2 serves as a receptor for the neurotransmitter-derived osas#9, and thus may function in both internal signaling and sensation of external signals. The TYRA-2/osas#9 signaling system thus provides an example for the evolution of an inter-organismal communication channel via co-option of a small-molecule signal and its cognate receptor. Author Summary Inter-organismal chemical communication relays information from one organism to another, and elicits specific behaviors in the receiving organism via downstream intra-organismal signaling pathways. Given the ancient origin of chemical communication, it seems plausible that evolution would favor re-use parts of the communication machinery. Previous work had identified the neurotransmitter-derived pheromone osas#9 which elicits avoidance behavior in conspecific C. elegans. Based on the assumption that additional components of neurotransmitter signaling may have been co-opted for perception of osas#9, we conducted a reverse genetic screen of neurotransmitter receptors and identified tyra-2, a tyramine/octopamine receptor as required for the osas#9 avoidance response. These results suggest that this signaling pathway has repurposed a neurotransmitter by “packaging” it into a pheromone molecule and retained the receptor of this neurotransmitter to sense this signal. Our findings reveal dual use of neurotransmitter communication machinery linking inter- and intra-organismal signaling.

Acute-stress impairs cytoprotective mechanisms through neural inhibition of the insulin pathway

Animals face both acute and long-term stressors that require behavioral and cellular defense strategies. How different stress responses are coordinated remains unclear. We show that the repeated induction of the C. elegans flight response reduces its ability to cope with environmental stressors. This acute-stress response triggers the activation of a single pair of neurons that release tyramine, the invertebrate analog of adrenaline/noradrenaline. Tyramine stimulates the DAF-2/Insulin/IGF-1 pathway through the activation of the adrenergic-like receptor TYRA-3 in the intestine. In contrast, exposure to long-term stressors reduces tyramine and DAF-2/IIS signaling, allowing the DAF-16/FOXO transcription factor to translocate to the nucleus and induce the transcription of cytoprotective genes. Tyramine signaling provides a state-dependent neural signal that controls the switch between acute and long-term stress responses to regulate the trade-off between behavioral and cellular defense mechanisms. These findings provide mechanistic insights how acute stress can impair the response to other environmental challenges.