• Structure of the nicotinic acetylcholine receptor
• Trapping the nicotinic acetylcholine receptor in the open state
• Photoaffinity labeling of the acetylcholine binding site
• Isolation of affinity-labeled polypeptide chains and fragments
• Identification of affinity-labeled amino acid residues
• Desensitization of the nicotinic acetylcholine receptor
• Functional states of the nicotinic acetylcholine receptor
• Ionotropic receptors in the cys-loop family
• Drugs that interact with GABA receptors and transporters
• Drugs that interact with glycine receptors and transporters

This family of ionotropic channels is the most important one for applied pharmacology, and it also contains the best-studied receptor molecule, namely, the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (NAR) is the most thoroughly studied member of the cys-loop family. It conducts cations, in a fairly nonselective way; Na+, K+ and Ca++ can all get across. It depolarizes the postsynaptic membrane.*

The structure shown here has been worked out with electron crystallography, which is a sort of hybrid between conventional crystallography and electron microscopy. The individual panels show the following:

A, B:

Electron micrographs of tubular synaptic membrane particles isolated fromTorpedo electric organs. The cross-section in B already shows the major structural features of the receptor. The formation of dense, regularly packed arrays as seen in A allows the collection of in-phase electron diffraction patterns, from which higher resolution contour maps can be constructed.

C, D:

Contour maps obtained by electron crystallography. In the side view (D), a constriction within the bilayer is clearly visible. The receptor has an intracellular domain with lateral openings.

E:

High resolution structural model, top view. The receptor consists of five subunits, each of which contributes one helix to the gate located at the level of the lipid bilayer.

F:

Side view of the structural model. Only one of the ? chains and the adjacent ? chain are shown. The tryptophan residue at position 149 of the ? chain is part of one of the two acetylcholine binding sites, which are located at the ?? and the ?? interfaces, respectively.

The subunit composition illustrated here applies to nicotinic receptors in neuromuscular synapses. Receptors at other anatomical sites differ in subunit composition, and some drugs discriminate between these different receptor types. Panels A–D courtesy of Nigel Unwin; E and F rendered from 2bg9.pdb.

Nigel Unwin managed to study the nicotinic acetylcholine receptor both in the open and the closed state. Now, considering that the open state has a lifetime of only a few milliseconds, how did he manage to prepare it, and then stabilize it so that it could be imaged? This slide explains his experimental setup.

1. The membrane vesicles containing the receptors, in 2D crystal arrangement and in the closed state, are mounted on an EM sample support and held in a forceps.
2. When the sample is dropped, it passes through a stream of vaporized acetylcholine. Binding of acetylcholine opens the channels. Immediately thereafter, the sample plunges straight into cold, liquid ethane, and the channels are shock-frozen in the open state.
3. Data acquisition for electron crystallography is performed at low temperatures, so as to preserve the receptor in its open state.

Rather brilliant, isn’t it? One of my favorite experiments.

The binding sites of acetylcholine in the receptor have been identified not by 3D-structural methods but by affinity labeling and protein chemistry. Multiple studies using different affinity probes have revealed different features of the binding sites; one such study is summarized here as an example.

The probe used in this experiment, 3H-TDBzcholine, contains a moiety that resembles acetylcholine (highlighted in blue), which steers it toward the acetylcholine binding site of the receptor. It is radioactively labeled and also photoactivatable, which enables it to covalently react with the receptor.

After allowing time for binding of the probe to the receptor, the probe is activated by exposing the sample to UV light. The energy of a photon absorbed by the probe’s aromatic moiety induces the release of nitrogen. This leaves behind a highly reactive carbene radical, which immediately reacts with amino acid residues in the vicinity. To identify the labeled amino acid residues, the receptor adduct is then fragmented in a stepwise fashion, as shown in the following slides.

In the first fractionation step, the hetero-oligomeric receptor was simply boiled in SDS and subjected to SDS-PAGE (left panel). The greatest extent of radiolabel incorporation was observed with the ? and the ? chain, indicating that these two chains surround the binding site.

In the second step, the labeled chains were fragmented by proteolysis. The protease used here, staphylococcal V8 protease, cleaves specifically after aspartic and glutamic acid residues. The fragments were separated by HPLC and identified by N-terminal sequencing. In the case of the ? chain (right panel), the radioactivity was associated with a fragment of 20 kDa that spans residues 174–339.

In both steps, control samples were included that had been exposed to 3H-TDBzcholine in the presence of unlabeled carbamoylcholine in excess. The unlabeled ligand displaced the3H-TDBzcholine from the acetylcholine binding site and largely suppressed labeling. This confirms that 3H-TDBzcholine indeed lodges within the regular acetylcholine binding site and not in some other, nonspecific binding crevice or pocket.

In the final stage of the experiment, the labeled proteolytic fragments were subjected to N-terminal sequencing through Edman degradation in order to identify the individual amino acid residues that had reacted with the label.

In the case of the labeled 20 kDa ?-chain fragment shown in the previous slide, residues 192–194 contained the most incorporated label, indicating that they are part of the acetylcholine binding site. Figures in both above slides were prepared from original data in [1].

As had been mentioned earlier (slide 2.5.5), the nicotinic acetylcholine receptor undergoes inactivation when bound to its ligand for prolonged periods of time. This property is important in the mode of action of the “depolarizing blocker” succinylcholine (slide6.15.1).

In the experiment illustrated here, acetylcholine was electrophoretically applied to frog muscle neuromuscular synapses, and the resulting postsynaptic potentials were recorded. Repetitive short acetylcholine stimuli evoke postsynaptic potentials of uniform amplitude. When continuous application starts, a strong depolarization occurs, which then declines even in the face of continued transmitter application. The response to superimposed short acetylcholine pulses also declines.

After continuous acetylcholine application is stopped, the response to short ACh pulses recovers over a period of several seconds. Drawn after an original figure in [2].

This slide shows a kinetic scheme for acetylcholine binding, receptor activation, and desensitization. When acetylcholine binds, the receptor initially opens, but then quickly inactivates. Opening is faster than inactivation, but the inactive conformation is more stable; therefore, after sufficient time, most receptor molecules accumulate in the refractory state.

As long as acetylcholine remains bound, the receptor remains inactive. Dissociation of acetylcholine is driven by cholinesterase, which depletes the transmitter by cleaving it to free choline and acetate. Once the transmitter has dissociated, the receptor reverts from the refractory state to the inactive one, from which it can again be activated by binding another acetylcholine molecule.

In contrast to a simple equilibrium reaction, which simply flickers back and forth between its two states, a unidirectional cycle like this one requires an input of energy. With the NAR, this energy is supplied by the hydrolysis of acetylcholine.

The nicotinic acetylcholine receptor occurs in the CNS, in the autonomic nervous system, and in neuromuscular synapses (motor endplates). We will see some drugs later, after having taken a look at the organization of the autonomic nervous system.

Inhibitors of the 5-HT₃ serotonin receptor are used to suppress emesis, particularly in cancer patients undergoing chemotherapy. The 5-HT₃ inhibitor ondansetron is shown in slide 6.12.2.

The GABA_A receptor and the glycine receptor are both chloride channels and therefore, when open, hyperpolarize the postsynaptic cells; they are the most important inhibitory neurotransmitter receptors in the brain. The GABA_A receptor is one of the preeminent drug targets in the CNS; agonists of this channel are used in conditions as diverse as epilepsy, psychoses, and narcosis. We have already seen several GABA_A agonists, such as diazepam and oxazepam (slide 4.2.2) as well as phenobarbital (slide 4.1.4) and thiopental (slide 3.6.9). Some more drugs that target GABA_A receptors are shown in slide 6.10.9.

The glycine receptor is less prominent as a drug target; some drugs interacting with it are shown in slide 6.10.10. Nevertheless, it has a crucial role in maintaining a proper balance of excitation and inhibition in the CNS, which is quite strikingly illustrated by the clinical manifestations that result when its function is disrupted. The clinical picture is known astetanus and consists in maximal, uncontrollable contraction of skeletal muscles, to the point where tendons snap and bones break.*

The GABA_A and GABA_C receptors are ligand-gated channels, whereas the GABA_Breceptors are GPCRs.

Isoflurane is an inhalation anesthetic, and etomidate is an intravenously applied anesthetic; both are GABA_A receptor agonists. Muscimol is an ingredient of fly agaric and a GABA_A and GABA_C receptor agonist. Pentylenetetrazol is a GABA_A antagonist used experimentally to induce seizures.

Baclofen is a GABA_B agonist that is used in the treatment of spasticity, for example in patients with multiple sclerosis. Tiagabine is an inhibitor of presynaptic GABA reuptake, which is used in the treatment of epilepsy.

Ivermectin is an allosteric agonist of the glycine receptor. Its main target, however, is a glutamate receptor/chloride channel that occurs in non-vertebrates including some human parasites, but not in humans; it is used in the treatment of parasite infections.

Strychnine is an alkaloid that acts as an antagonist of the glycine receptor. Once upon a time, it was used as a stimulant, as well as an important ingredient of murder novels. The symptoms of strychnine poisoning resemble those of tetanus. Wikipedia’s page on this subject is well worth a read.

Org 29535 is an inhibitor of glycine reuptake that is being investigated for the treatment of psychosis and addiction.