Thompson AJ et al. (2014), Phenylalanine in the pore of the Erwinia ligand...

XB-ART-49682

Biochemistry 2014 Oct 07;5339:6183-8. doi: 10.1021/bi5008035.

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Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

Thompson AJ , Alqazzaz M , Price KL , Weston DA , Lummis SC .

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The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

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Species referenced: Xenopus laevis
Genes referenced: gabarap pxn

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	Figure 1. Alignment of M2 channel-lining residues for ELIC with eukaryotic Cys-loop receptors and a cartoon showing two of the five pore-forming transmembrane α-helices, highlighting the location of F16′ and L9′. As is common for these receptors, a prime notation is used to facilitate comparison between different subunits, with 0′ being the conserved charged residue at the start of M2. Accession numbers are P0C7B7 for ELIC, P46098 for 5-HT3A, P02708 for nACh α1, P23415 for Gly α1, and P14867 for GABAA α1.
	Figure 2. Example traces of PXN inhibition of wild-type and mutant ELIC. (A) Co-application of GABA EC50 with 100 μM PXN inhibits ∼50% of the wild-type and F16′W responses but completely abolishes the F16′S response. (B) Increasing concentrations of PXN sequentially diminish the GABA-elicited response (here 0, 10, 100, and 300 μM PXN with 0.8 mM GABA). Data such as these were used to create the inhibition curves shown in Figure 3.
	Figure 3. PXN inhibition of wild-type and mutant ELIC. (A) Inhibition curves of wild-type and F16′ mutant ELIC responses. (B) Inhibition curves of wild-type and T6′ mutant ELIC responses. Data are means ± SEM (n = 3–6). Parameters obtained from these curves are listed in Tables 2 and 3.
	Figure 4. pIC50 vs side chain volume. Volumes are calculated from the surface area of the side chain.26
	Figure 5. Overlay of 10 docked poses for PXN in the ELIC pore. The channel is seen from the side, and the ligands are located above F16′ (left) or close to the residues at position 6′ (right).
	Figure 6. Example docked pose for PXN in the ELIC channel. PXN is located close to the Thr residues at position 6′, where it is stabilized by hydrogen bonds. The channel is seen from the top looking down toward the cell interior.