Cys-Loop receptors Flashcards
Basic structure
Determined by 3D electron microscopy
Pentameric LGICs
5 subunits, each with 4 TM helices M1-4
M2 forms the pore, M1 and 3 surround, M4 only loosely associated at the outside
Protrudes 60 amstrongs intracellularly, 40 extracellularly (so shorter than glutamate receptors)
The only conserved residues /within/ agonist binding loops are a tryptophan in Loop A and a Trp-X-Proline in loop D, thought to contribute to structural integrity in the 5-HT3 receptor. However, there is almost always an ‘aromatic box’ in the binding site, which surrounds the charged amine in ligands, though exact positions of its contributors vary. The aromatic residue on loop B that’s part of this box (usually TrpB) is the one that forms the cation-pi interaction
Location and role
Located at the synapse only, in the centre of a bullseye with GPCRs around the outside (e.g. glycine receptor clustering is mediated by gephyrin)
GABA<a> and GlyRs mediate fast inhibitory transmission
nAChR and 5-HT3 mediate fast excitatory transmission</a>
In invertebrates only, GluCl (glutamate gated chloride channel) mediates fast inhibitory transmission - ivermectin is an allosteric agonist, activates GluCl at nM concentrations, so is used as an antiparasitic. But careful - activates other pLGICs and P2X-like receptors at higher concentrations.
GLIC/ELIC thought to be important for photosynthesis. Both cation-selective, both transiently gate open in response to agonist binding - agonist for GLIC is protons, for ELIC is primary amines like cysteamine and GABA</a>
Difficulties determining crystal structure
Much the same as for GPCRs - proteins are unstable in isolation, so needed stabilising with antibodies etc
X-ray crystallography damaged samples, which often weren’t big to begin with
Before we found prokaryotic channels, it was hard to work with eukaryotic cell lines, and to express enough protein, and get pure samples
ICD in particular is hard to crystallise, so normally got chopped off
History of structural studies
Muscle nAChR was the first LGIC to be cloned (including glutamate receptors and P2X-like receptors) Structures revealed: AChBP in 2001 nAChR closed in 2005 ELIC closed 2008 GLIC open 2009 GluCl +IVM/FAb open 2011 2014 gave us 4 structures 2015 4 more now the focus is on finding these receptors in other conformations
What did we know about nAChR before AChBP crystal structure was found?
Water soluble LBDs of nAChR had been produced, but not in sufficient quantities for high resolution crystal structure
Biochemical and electrophysiological data for nAChR (it was the first protein to yield single channel patch clamp recordings)
Gross structure (Brisson and Unwin 1985 determined its quaternary structure as pentameric, with an hourglass shape, by homogenising Torpedo electric organ and leaving receptors out to form tubes. Pentameric computer models fit best)
High resolution EM had shown ACh binding pockets surrounded by beta sheets, which fit with circular dichroism measurements
No atomic structural info.
Identifying binding site
Kao et al 1984 - used affinity reagent labelling (with competitive antagonists) to identify cysteine in alpha subunit necessary for ACh binding
Affinity reagents bind beta and gamma subunits to a lesser extent. This asymmetry suggests the alpha subunit has a ‘principal component’ and the gamma/delta a ‘complementary component’.
Binding pocket for ACh was only found in pairwise expression of the alpha subunit with either beta or gamma - any other pair or single expression did not give the pocket
Further affinity labelling, proteolysis and Edman degradation experiments determined three loops on the alpha subunit (A, B and C) and three on the complementary subunit (D, E and F) that were involved in the binding
Sequence comparison showed that the labelled residues in loops A-D were highly conserved, F mostly conserved, and E highly variable.
Intracellular domain
We don’t know much about it, because it’s hard to crystallise
Consists of M3-M4 loop
Absent in GLIC/ELIC
Deletion studies show it’s not required for expression, and cutting it off doesn’t prevent basic function of receptor, but is important in localisation to synapse
Contains ‘portals’ at the side to allow ions to stream out parallel to membrane, perhaps enhancing effect on membrane potential
Amino acids in the circumference of these portals affect conductance - in 5-HT3A receptors, there are arginines in 4 specific places in the M3-M4 loop. In 5-HT3B receptors, there are neutral or negatively charged AAs here. This explains 5-HT3AR 200-fold lower conductance than nAChR. Expression of the AAs from the B subunit rescues conductance
GLIC doesn’t have an ICD, and isn’t inhibited by Ric-3 (which is a chaperone protein that interferes with assembly and function of 5-HT3R). If you give GLIC the 5-HT3R ICD, it is inhibited by Ric-3!
Insights from chimaera
Eisele et al 1991 - nAChR is calcium permeable, and permeability response to ACh binding is potentiated by calcium. 5-HT3 is calcium impermeable, and blocked by calcium. To determine which parts of the structure conferred these characteristics, they made a chimaera combining n-terminal of alpha7 and TM domain + c-terminus of 5-HT3. They found that properties like permeability and ligand binding were determined by the receptor that the TM domain or ECD came from, respectively. However, kinetics of current onset and desensitisation, fast in alpha7 WT and slow in 5-HT3 WT, were intermediate in the chimaera, suggesting interactions between ECD and TM are responsible.
GLIC doesn’t have an ICD, and isn’t inhibited by Ric-3 (which is a chaperone protein that interferes with assembly and function of 5-HT3R). If you give GLIC the 5-HT3R ICD, it is inhibited by Ric-3! This suggests ICD is important for transport to membrane.
Insights from AChBP
Produced by glial cells in molluscs
Pharmacological profile most similar to alpha7
AChBP was useful because it’s structurally and functionally homologous to the ligand binding domain of nAChR, but it’s water-soluble, so easier to use crystallography. X-ray crystal structure
Brecj et al 2001:
Ten beta sheets in each protomer, forming a beta sandwich reminiscent of a Ig fold structure, with a hairpin, but with an extra strand. Compared with the classic Ig-fold, the B-sheets are rotated around one another, forming a hydrophobic core.
There was a different cavity at each interface between the subunits. It was buried from solvent and position close to the edge of the pentameric ring. They matched the expected position of ligand binding sites from labelling and EM studies of the nAChR.
The 6 loop system was confirmed, and relative location of conserved residues revealed.
They also found HEPES buffer molecule bound in a ligand binding pocket, confirming the suspected cation-pi interaction
Brams et al 2011 - compared crystal structures of 30 AChBPs in complex with different ligands. Identified Loop C contraction.
Remember it may differ in detail though!
What is a cation-pi interaction? How was it discovered in pLGICs?
A stabilising interaction between the face of an aromatic residue and a cation
Broadly used to stabilise secondary structure and receptor-ligand binding.
Comparable strength to a hydrogen bond or ion pair
There are six times as many Trp residues in the binding pocket as you’d expect from other known proteins. This suggested non-ionic bonds like the cation-pi interaction.
Zhong et al 1998 - showed a correlation between quantum mechanical predictions from a cation-pi model, and EC50 values from in vivo nonsense suppression method of unnatural amino acid incorporation, adding Fluorines to a tryptophan residue in loop B. The more F added, the weaker the cation-pi interaction (because it destabilises the electrostatic ring and the higher the EC50). Also, adding a tethered a quaternary ammonium group to this tryptophan produces a constitutively active receptor.
Xiu et al 2009 - cation-pi interaction between nicotine and brain nAChR can form because there’s a lysine at 153 that hydrogen bonds to loop B and lets nicotine in. In the muscle nAChR it’s a glycine, nicotine can’t get in, no muscle spasms when smoking
H bonds via water molecules
In 5-HT3R: When a conserved serine is swapped to a threonine (which still has an -OH group), there’s no effect on affinity. When it’s swapped to an alanine (no -OH group), affinity drops. This H bond is via water, and with backbone groups rather than R groups or ligand itself. One of the sites was on the opposite side of a beta sheet to ligand entry, so must be an intra-molecular thing rather than direct ligand affinity thing.
In AChBP there’s another conserved water molecule that forms bonds between partial agonists and hydrophobic residues on the complementary side of the binding pocket. Carbamylcholine (a full agonist) binds directly, so it was proposed that this indirect binding is indicative of partial agonists.
BUT Tavares et al 2012 found no intervening water molecule in varenicicline-bound structures. Possibly observing an H bond doesn’t mean it’s important, or ligands bind in different poses in AChBP vs nAChR.
Other interactions, loop D and E
Conserved hydrophobic interaction between loo D and varenicicline in AChBP
Loss of aromatic residues in loop D and of leucine in loop E of alpha4beta2 nAChR eliminates difference between efficacy of varenicicline and full agonists, and eliminates varenicicline-mediated desensitisation
Changing a tyrosine residue at 153 in 5-HT3R to phenylalanine had no affect, but changing it to serine reduced affinity, suggesting aromatic interaction
Pore structure - finding the pore, creating the numbering system
Imoto et al 1986 - chimaerae of calf and torpedo delta subunits - all you had to change to swap conductances was M2
Models of pore as five kinked M2 helices were generated by molecular dynamics simulations incorporating restraints from the cryo-EM structures available at the time, from Unwin’s work.
Blocking mechanism of agents such as chlorpromazine was investigated to reveal a conserved lysine or arginine at the narrowest part of the pore, labelled 0. There’s also a conserved leucine at 9, right in the middle of the pore, which forms a hydrophobic barrier that extends to 13’valine and stops ions going through the closed pore (would require dehydration).
Every third or fourth AA faces the pore, and can be compared between receptors.
[the TM helices are the target for anaesthetics, steroids, alcohols]
Pore structure - finding selectivity filters, and more detailed structure
Corringer et al used mutagenesis to find three rings of selectivity -negatively charged residues at the extracellular side, Glu at -1prime, and Glu/Asp at -4 prime. The intermediate ring is the most important, because whilst mutations in the other rings can affect conductance gradually, a mutation in the intermediate causes a v sudden drop in g.
Akabas et al 1994 mutated every residue in and flanking M2 of the alpha subunit to cysteine, one at a time. The ones that reacted with MTSEA (applied extracellularly) were accessible. They found 5, so at least this many are facing the pore - this was consistent with a helic structure. ACh subtly changed accessibilities, suggesting a change in shape of the pore.
–Caveat: in the 5-HT3 receptor, MTS caused an agonist-independent current when serine of threonine was mutated to cysteine, so it may be altering conformation and thus giving false positives-negatives about accessibility. –
A series of mutations in M2 were analysed used the rate-equilibrium free energy relationship to determine which position of each residue was temporally closer to the open or closed position. Determined that the extracellular half of the pore undergoes conformational change before the intracellular half, and indeed may be in different conformations whilst the pore is functionally closed
Selectivity transition mutant
Swap -1 glutamate (intermediate ring) for alanine, add proline at -1’’, change 13’ valine to threonine, then you swap to anion-selective
You know it’s anion selective now because swapping all the external Cl- for a nonconducting anion you get a big change in reversal potential
This same change works for 5-HT3R and for nAChR
