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Cell Mol Life Sci
2024 Aug 07;811:332. doi: 10.1007/s00018-024-05374-1.
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Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.
Mukhtasimova N
,
Bouzat C
,
Sine SM
.
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Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drug modulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium may alter this balance and modulate the ability of PNU to potentiate α7.
NS031744 National Institute of Health, PGI 24/B298 Universidad Nacional del Sur, PICT 2020 00936 Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, PIP 11220200102356 Consejo Nacional de Investigaciones Científicas y Técnicas, R01 NS031744 NINDS NIH HHS , R37 NS031744 NINDS NIH HHS
Fig. 1. Structure of α7 with bound agonist, calcium and PNU. Cryo-EM structure of α7 (PDB: 8V8A) showing the protein backbone (blue lines), the agonist epibatidine (green sticks), PNU (magenta sticks) and calcium (red spheres). Views from the top and side are shown. Horizontal lines indicate the approximate location of the cell membrane. The intracellular domain is omitted for clarity
Fig. 2 Please move this figure forward by one printed page in the PDF. PNU-potentiation of concatemeric (α7)5 depends on the number of agonist binding sites and divalent cations. Single-channel currents were recorded from Bosc-23 cells expressing concatemeric receptors with (a) five agonist binding sites, (α7)5, or (b) one binding site, (α7)1(α7Y188V)4, without or with Ca2+ and Mg2+ or with only Ca2+, Mg2+ or Sr2+ as the divalent cation. Schematic diagrams depict wild type α7 subunits in white and subunits with the mutation Y188V in red. For each receptor and condition, representative traces of single channel currents and the corresponding cluster duration histograms fitted by multiple exponentials are shown. Single channel currents were elicited by 100 µM ACh in the presence of 10 µM PNU at a membrane potential of − 100 mV, displayed with a Gaussian filter of 5 kHz. Channel openings are upward deflections from baseline
Fig. 3. Electrical fingerprinting strategy to distinguish single channel currents from receptors with different subunit stoichiometries. (a) Close up view of a region of α7 encompassing the intracellular domain, with the protein backbone in blue lines and residues mutated to generate the LC form of α7 in red sticks. For clarity only three subunits that bracket two intracellular portals are shown; one portal is visible as the open space flanked by red sticks to the left. For reference the location of the intracellular membrane leaflet is indicated by horizontal lines. (b) Segments from a representative recording from a cell co-expressing HC and LC subunits with Ca2+. ACh, 100 µM; PNU, 10 µM; membrane potential, − 100 mV; Gaussian filter, 5 kHz. (c) Plot of the mean current amplitude of each amplitude class against the inferred number of HC subunits per pentamer; HC subunits are depicted in grey with black outline and LC subunits in white with red outline. The data correspond to the mean ± SD of 5 independent recordings. (d) Plot of current amplitude of each cluster against its duration from a representative recording. The plot shows six stripes of points, each corresponding to a different amplitude class and stoichiometry of HC and LC subunits
Fig. 4. PNU-potentiation of α7 nAChRs composed of unlinked subunits depends on the number of agonist binding sites and calcium. Single channel currents were recorded from cells co-expressing HC-Y188V and LC subunits without (a) or with (b) Ca2+. ACh: 100 µM, PNU: 10 µM, membrane potential: −100 mV, Gaussian filter, 5 kHz. For each condition, the panels from left to right show: Representative traces of single channel currents of the highest amplitude class corresponding to receptors with one agonist binding site without (a) and with (b) Ca2+. In the traces in panel (a), brief openings with reduced amplitude are not fully resolved and thus are not included in any amplitude class (see Materials and Methods). Plots of current amplitude of each cluster against its duration from a representative recording without (a) or with (b) Ca2+. Each plot shows five discrete amplitude classes of clusters corresponding to receptors with different subunit stoichiometries. The highest amplitude class corresponds to receptors with one agonist binding site comprised of four HC-Y188V subunits and one LC subunit; Mean cluster durations for receptors with the indicated numbers of LC subunits in the absence (white bars, a) or presence (grey bars, b) of Ca2+. Each symbol corresponds to a recording from a different membrane patch. The data correspond to the mean ± SD of 5–6 independent experiments recorded from different cells. -Plots of the fraction of clusters with greater than N re-openings against the number of re-openings per cluster in the absence (a) or presence (b) of Ca2+. Data correspond to receptors containing either one (black) or two (red) LC subunits, each forming an ACh binding site, for each experimental condition. Data are fitted by a single or double exponential decay, with the fitted parameters in Table 1. Each plot includes data from 5 recordings for each condition
Fig. 5. Contributions of key anionic residues to PNU potentiation. (a) Close up view of the structural region of α7 that forms a calcium binding site, with the protein backbone shown as blue lines, calcium as a red ball, and flanking electron-rich residues as sticks (PDB: 7KOQ). Single channel currents were recorded either without or with Ca2+ from cells expressing a HC-E45V (b) or HC-D44N (c) subunit together with the LC subunit. ACh: 100 µM, PNU: 10 µM, membrane potential: 100 mV, Gaussian filter, 5 kHz. For both mutant receptors, the panels from left to right show: representative traces of currents of the highest amplitude class corresponding to receptors with five mutant HC subunits (black circle, zero intact calcium sites), currents from receptors with four mutant HC subunits and one LC subunit (red circle, one intact calcium site), in the absence or presence of Ca2+. Bar graphs showing the mean cluster duration for each amplitude class corresponding to receptors with the indicated numbers of unaltered calcium binding sites recorded with (grey bars) or without Ca2+ (white bars). The data correspond to the mean ± SD of 5–6 independent experiments recorded from different cells. Plots of the fraction of clusters with greater than N re-openings against the number of re-openings per cluster without (top) or with (bottom) Ca2+. Data correspond to receptors containing zero (black line) or one (red line) unaltered calcium binding site. Each plot includes data from 5 recordings for each condition. Data were fitted by either a single or double exponential decay, with the fitted parameters in Table 2
Fig. 6. Interdependence between agonist and calcium binding sites in PNU potentiation. Receptors were formed by co-expressing the HC-Y188V subunit with an LC subunit (a) or the HC-Y188V subunit with an LC subunit carrying a mutation of the indicated anionic residue (b–e). Single channel currents were recorded with 100 µM ACh and 10 µM PNU without (left) or with (right) Ca2+. The bar graphs show the mean cluster durations ± SD for receptors with the indicated ratios of agonist to intact calcium binding sites without (white bars) or with (grey bars) Ca2+ (n = 5–7 independent recordings for each condition from different cells). The re-opening plots show the fraction of clusters with greater than N re-openings against the number of re-openings per cluster in either the absence or presence of Ca2+. For each curve, the color represents the stoichiometry of the receptor indicated by the black, blue or red circle in the corresponding bar graph. Each plot includes data from 5 to 7 recordings for each condition. Data were fitted by a single or double exponential decay, with the fitted parameters in Table 3
