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Front Physiol
2022 Jan 01;13:850782. doi: 10.3389/fphys.2022.850782.
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Mutations of the nACh Receptor M4 Helix Reveal Different Phenotypes in Different Expression Systems: Could Lipids be Responsible?
Mesoy SM
,
Bridgland-Taylor M
,
Lummis SCR
.
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The role of the outermost helix (M4) in the pentameric ligand-gated ion channel (pLGIC) family is currently not fully understood. It is known that M4 is important for receptor assembly, possibly via interactions with neighboring M1 and M3 helices. M4 can also transmit information on the lipid content of the membrane to the gating mechanism, and it may form a link to the extracellular domain via the Cys-loop. Our previous study examining the α4β2 nACh receptor M4 helix using HEK cells indicated M4 here is more sensitive to change than those of other pLGIC. Many of these other studies, however, were performed in Xenopus oocytes. Here we examine the nine previously identified nonfunctional α4β2 nACh receptor M4 mutant receptors using this system. The data reveal that seven of these mutant receptors do function when expressed in oocytes, with only 2, the conserved Asp at the intracellular end of M4 and a Phe in the center, having a similar phenotype (nonfunctional) in both HEK cells and oocytes. The oocyte data are more consistent with studies in other pLGIC and demonstrate the importance of the expression system used. Of the many differences between these two expression systems, we suggest that the different lipid content of the plasma membrane is a possible candidate for explaining these discrepancies.
FIGURE 1. Sequence alignment of some example pLGIC M4 helices. Uniprot numbers are, in order, P09483, P12390, P02708, Q05941, Q8K1F4, P07727, P50572, Q7NDN8, and P0C7B7. Where residue types are predominantly conserved or identical they are coloured grey or dark grey, respectively. Residue numbers for the α4 and β2 nAChR subunits are shown below (α4/β2), along with the positional numbering system used here, where the conserved Asp at the intracellular end of M4 defines position 4.0.
FIGURE 2. Characterization of α4β2 nACh receptors in Xenopus oocytes. (A) Typical currents elicited by 1 μM nicotine application to receptors expressed in oocytes. Scale bars are 500nA and 10s. (B) Concentration-response curves. Data = mean ± SEM, n = 3â4. Parameters derived from these data are shown in Table 1.
FIGURE 3. X-ray structure (5kxi) of human α4β2 nACh receptors in the closed state showing α4 (brown) and β2 (blue) transmembrane helices M1, M3, and M4. Selected M4 residues probed in this study and their possible aromatic interaction partners on M1 and M3 are shown as sticks. Distances along dashed lines in à .
FIGURE 4. Concentration-response curves of α4β2 nACh receptors expressed in HEK cells. Data = mean ± SEM, n = 4. Functional parameters from these curves are shown in Table 2. Inset: typical responses to 300 nM nicotine applied at *. Scale bars = 40s and 50 arbitrary fluorescent units.
FIGURE 5. Specific binding of 3H-epibatidine relative to WT in single and double mutant nACh receptors in Table 3. Only the WT value is significantly different from untransfected (UT) cells. Data = mean ± SEM, n = 3â5. + indicates coexpression with chaperones.
FIGURE 6. Cholesterol binding pockets. Two cholesterol moieties (cyan) binding to the α4 (brown) and β2 (blue) subunits in the nACh receptor (6CNJ). Shown in sticks are residues on M4 and M1 that may interact with cholesterol.
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