Lummis SCR and Dougherty DA (2022), Expression of Mutant Glycine Receptors in Xenop...

XB-ART-59425

Membranes (Basel) 2022 Oct 19;1210:. doi: 10.3390/membranes12101012.

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Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues.

Lummis SCR , Dougherty DA .

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Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a structural change that is transduced to the TMD to open the channel. Molecular details of this process are emerging, but a comprehensive understanding is still lacking. Proline (Pro) is one amino acid that has attracted much interest; its unusual features generate bends in loops and kinks and bulges in helices, which can be essential for function in some pLGICs. Here, we explore the roles of four conserved Pros in the glycine receptor (GlyR), creating substitutions with canonical and noncanonical amino acids, characterizing them using two electrode voltage clamp electrophysiology in Xenopus oocytes, and interpreting changes in receptor parameters using structural data from the open and closed states of the receptor. The data reveal that for efficient function, the Pro in the α1β1 loop is needed to create a turn and to be the correct size and shape to interact with nearby residues; the peptide bond of the Pro in the Cys-loop requires the cis conformation; and the Pros in loop A and M1 allow efficient function because of their reduced hydrogen bonding capacity. These data are broadly consistent with data from other pLGICs, and therefore likely represent the important features of these Pros in all members of the family.

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Genes referenced: ecd ttn

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References [+] :

Baker, Proline Residues Contribute to Efficient GABAp Receptor Function. 2020, Pubmed, Xenbase

	Figure 1. Cartoon of a GlyR α1 subunit showing the location of Pros examined in this study. P30 and P96 are in the ECD, P146 is at the ECD/TM interface, and P230 is on M1 in the TM domain.
	Figure 2. Concentration response curves for WT and mutant receptors derived from maximal responses (see Figure 3 for examples). Data = mean ± SEM, n = 5. Parameters derived from these data are shown in Table 1.
	Figure 3. Typical Gly (1 mM) and Tau (10 mM)-induced traces for WT and mutant receptors expressed in oocytes and clamped at −60 mV. Bars indicate agonist application. The different shapes of the responses suggest the mutations may alter receptor kinetics (e.g., desensitization, association and/or dissociation rates).
	Figure 4. Concentration response curves for WT and mutant receptors T/G = ITau/I max Gly. Data = mean ± SEM, n = 5. Parameters derived from these data are shown in Table 1.
	Figure 5. Concentration response curves for WT and mutant receptors substituted with Pro analogues. Data = mean ± SEM, n = 4–6. Parameters derived from these data are shown in Table 2.
	Figure 6. Concentration response curves for GlyR expressed in oocytes where P146 was substituted with Pro or Pro analogues using nonsense suppression. T/G = ITau/I maxGly. Parameters derived from these data are shown in Table 3. Data are mean ± SEM, n = 3–4.
	Figure 7. P30 is located at a tight turn in the α1β1 loop and its substitution could alter this turn and/or interactions with or between adjacent residues such as the conserved W68, D70 or R72 in loop D, W94 in loop A or Y24 in the α1β1 loop.
	Figure 8. P96 faces the interior of the subunit ECD and its distance from Q66 differs slightly in the open (green) and closed (blue) GlyR structures.
	Figure 9. (A) Relationship between EC50s and cis-trans preferences for Pro and analogues at position 146. All values are relative to Pro. R2 = 0.96, slope = significantly non zero p = 0.004. Cis-trans (c-t) values for Pip and CFP from Limapichat et al. [24], for TFP from Pandey et al. [25], and for 2MeP from Kang & Park [26]). (B) Relationship between maximal Tau /Gly induced responses (Figure 4) and % cis (from Limapichat et al. [24]); R2 = 0.92, slope = significantly non zero p = 0.04.
	Figure 10. The M1 helix has different structures in the open (green) and closed (blue) states of the GlyR in the region just above P230, and there is also a difference in the adjacent part of M2. The closest M2 residue, Q266, is 2.8Å from P230 in the former and 3.2Å in the latter.