Crnjar A et al. (2021), A Single Mutation in the Outer Lipid-Facing Hel...

XB-ART-58115

Front Mol Biosci 2021 Apr 13;8:644720. doi: 10.3389/fmolb.2021.644720.

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A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.

Crnjar A , Mesoy SM , Lummis SCR , Molteni C .

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Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC-the 5-HT3A receptor-unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

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Species referenced: Xenopus laevis

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	Figure 1. (A) Model of the open structure of the 5-HT3A receptor (PDB ID 6DG8, Basak et al., 2018a) embedded in a lipid membrane. The five subunits are colored in red, yellow, green, blue and orange. (B) One subunit of the 5-HT3A receptor; M1: yellow, M2: blue, M3: red, M4: purple, Cys-loop: orange. The atoms of 5-HT and residue Y441 are shown as spheres. The two possible paths of Y441A action are shown in light blue (radial) and red (vertical). (C) View orthogonal to the channel axis of the transmembrane domain of the 5-HT3A receptor.
	Figure 2. RMSF of backbone atoms of the M4 amino acids, calculated for R01-400 for the WTR (blue) and the MR (yellow).
	Figure 3. (A) Average dynamic correlation of residue 441 backbone atoms with those of residues 448 and 459, for the WTR (blue) and the MR (yellow) in the two replicas R0 and R1. Subscripts denote the five subunits. (B) Position of residues 441, 448, and 459 in the protein.
	Figure 4. Hydrogen bonds of M4 tip residues (I458, W459, H460, Y461, S462), calculated over R01-400 for the WTR (blue) and the MR (yellow).
	Figure 5. π-π interactions (F438-F424,441-F242,Y448-Y286,W456-F144,W459-F144) and anion-π interactions (441-D238) of different amino-acids of M4, calculated over R01-400 for the WTR (blue) and the MR (yellow).
	Figure 6. (A,B) Typical fluorescent responses (F in arbitrary units, AU) to addition of 5-HT at 20 s to HEK293 cells expressing 5-HT3A receptors, using a membrane potential sensitive dye. (A) WT, (B) C290A. (C) 5-HT concentration-response curves of mutant receptors in HEK293 cells. For D238A F is compared to WT Fmax. Data is mean ± standard error of the mean (SEM), n ≥ 3. (D) Molecular dynamics snapshot showing Y441 in the M4 helix together with possible interaction partners. M1: yellow, M3: red, M4: purple.
	Figure 7. Hydrogen bonds of residue 441 (left) and D238 (right) with any other protein residue, calculated over R01-400 for the WTR (blue) and the MR (yellow).
	Figure 8. (A) Molecular dynamics snapshots showing how Y441 might help maintain the hydrogen bonding between D238 and K255 in the WTR, while in the MR these two residues are less likely to form interactions. M1: yellow, M2: blue, M4: purple. (B) Distributions of the distance between Cγ of D238 and the terminal nitrogen of K255, in the two models. Thick lines: averages; shaded area: error.
	Figure 9. Concentration-response curves of 5-HT3A receptors in Xenopus oocytes. Data is mean ± SEM, n≥3. Inset: typical current recordings at 3 μM 5-HT; scale bars are 20 s and 2 μA.
	Figure 10. pEC50 relative to WT for mutants expressed in HEK293 cells and Xenopus oocytes, from Tables 1–3. Data is mean ± SEM, n ≥ 3; values less than 1 indicate loss-of-function, and values greater than 1 indicate gain-of-function.
	Figure 11. Proximity lifetime distributions of any phospholipid (top) and of any cholesterol (bottom) in close proximity to residue 441 in the WTR (blue) and in the MR (yellow) for replicas R0 (left) and R1 (right). The results are shown for residence times larger than 5 ns.
	Figure 12. Snapshots from the MD simulations showing some of the lipids that form hydrogen bonds with residues 441 and/or 238 in the WTR (A) and MR (B–D). These are discussed in the text. M1: yellow, M2: blue, M4: purple.