We begin with the premise of topographically distinct binding sites for agonist, calcium and PNU, each present in five copies per α7 pentamer (Fig. 1). Our goal is to investigate the functional interplay between the agonist and calcium binding sites and PNU potentiation. The investigations toward this goal are divided into three sections, each addressing a different facet of PNU potentiation using single channel patch clamp recordings in the presence or absence of calcium.
Fig. 1Structure 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
The first section employs either concatemeric receptors or an electrical fingerprinting approach to manipulate the number of agonist binding sites while monitoring PNU potentiation. In the second section, we generate mutants of key residues at an inter-domain calcium binding site, manipulate the number of copies of each mutant per receptor, and monitor PNU potentiation. The third section manipulates both the number of agonist and intact calcium binding sites and monitors PNU potentiation.
PNU potentiation depends on the number of agonist binding sites and divalent cationsPrevious studies demonstrated that in the presence of ACh alone, α7 activates as brief submillisecond channel openings flanked by long closings, or occasionally, several openings in quick succession [19, 25]. However, in the presence of ACh and PNU channel openings become prolonged and form clusters of many openings lasting from hundreds of milliseconds to several seconds [22, 30].
To investigate the interplay between agonist, PNU, and calcium binding sites, we employed a concatemeric α7 receptor composed of five α7 subunits connected by short polypeptide linkers. This construct, designated (α7)5, mimics the wild-type α7 receptor in its response to ACh and potentiation by PNU [26]. Using (α7)5 as a template, we introduced the mutation Y188V into one or more subunits to manipulate the number of agonist binding sites, with precise control over both the number of mutant subunits and their positions within the pentamer. Previous studies showed that substituting threonine for Y188, a residue essential for agonist binding, abolished agonist-elicited macroscopic or single channel currents and binding of α-bungarotoxin [31]. These observations showed that substituting threonine for Y188 disabled the agonist binding site. Here we substituted valine for Y188 to mirror the geometry of threonine and eliminate potential interactions with polar groups within ACh. Hereafter, a subunit that contains the native Y188 is assumed to form a functional agonist binding site, whereas a subunit that contains the Y188V mutation is assumed to form a disabled binding site.
We expressed (α7)5 in Bosc 23 cells, a clonal cell line derived from HEK cells, and recorded single channel currents elicited by ACh in the presence of PNU either without calcium or with physiological concentrations of calcium and magnesium; hereafter these two solutions are designated either without or with calcium, respectively. To maximize PNU occupancy a concentration of 10 µM PNU was used throughout. The recordings reveal long clusters of potentiated channel openings either without or with calcium (Fig. 2a). Additionally, a modest decrease in the unitary current amplitude is observed with calcium, as described previously [19].
Fig. 2 Please move this figure forward by one printed page in the PDFPNU-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
To quantify the mean durations of the clusters, we define a cluster as a series of channel openings separated by brief closings shorter than a defined critical time, tcrit, as outlined in Materials and Methods. We then constructed histograms of cluster durations and fitted multiple exponential components to the histograms; the exponential component with the longest mean duration corresponds to clusters of fully potentiatiated channel openings. The results show that the cluster duration histograms are similar without or with calcium (Fig. 2a), indicating that in receptors with five agonist binding sites, PNU potentiation does not depend on calcium.
However, in concatemeric receptors with four of the five agonist binding sites inactivated by the mutation Y188V, designated (α7)1(α7Y188V)4, potentiated channel openings are observed with but not without calcium (Fig. 2b). Without calcium, channel openings appear as single brief spikes flanked by long closings, leading to a marked shift of the cluster duration histogram by several orders of magnitude toward shorter durations (Fig. 2b). Thus, in α7 receptors with only one agonist binding site calcium is pivotal in conferring potentiation by PNU.
To assess divalent cation specificity for PNU potentiation, we individually tested calcium, magnesium, and strontium on the (α7)1(α7Y188V)4 concatemer. The recordings reveal long clusters of channel openings in the presence of each divalent cation, with each cluster duration histogram showing a major exponential component with prolonged mean duration corresponding to potentiated channel openings (Fig. 2b). Thus, for α7 receptors with a single agonist binding site divalent cations of differing sizes enable PNU potentiation.
Next we increased the number of agonist binding sites within the the concatemeric receptor and monitored PNU potentiation. In contrast to receptors with a single agonist binding site, receptors with two or three sites, designated (α7)2(α7Y188V)3 and (α7)3(α7Y188V)2, respectively, exhibit potentiated channel openings either without or with calcium, as shown by long clusters of channel openings and a major exponential component of clusters with prolonged mean duration (Fig. S2). Thus increasing the number of agonist binding sites beyond one overcomes the requirement of calcium for PNU potentiation. Given the novel interdependence between the number of agonist binding sites, PNU potentiation and calcium, we sought to determine the molecular and mechanistic underpinnings of this phenomenon.
Studies of receptors with unlinked subunits-The preceding studies manipulated the number of agonist binding sites using concatemeric receptors in which the subunits were connected via short peptide linkers. To exclude possible influences from the peptide linkers, we employ an electrical fingerprinting approach. This approach involves co-expression of two different unlinked subunit types (Fig. 3a): one with arginine substitutions for anionic or polar residues flanking intracellular portals, designated LC for low conductance, and another retaining the original anionic or polar residues, designated HC for high conductance [25, 26, 30,31,32]. Located within the intracellular domain, these portals are far from the calcium binding sites near the extracellular membrane leaflet, and thus are not expected to impact binding of calcium.
Fig. 3Electrical 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
Recordings from control receptors, generated from HC and LC subunit mixtures, reveal long clusters of potentiated channel openings. However, the current amplitudes of the openings exhibit discrete, evenly spaced values owing to variations in the stoichiometric ratio of HC to LC subunits within individual pentameric receptors (Fig. 3b, c). Accordingly, a plot of current amplitude of each cluster against its duration reveals six horizontal stripes of points, corresponding to receptors with zero to five HC subunits (Fig. 3d). Notably, each horizontal stripe spans a similar brief to long time range, indicating cluster duration is independent of the stoichiometric ratio of HC to LC subunits.
To manipulate the number of agonist binding sites per receptor, we co-expressed HC-Y188V and LC subunits and recorded single channel currents elicited by ACh in the presence of PNU, either without or with calcium. In this experiment, receptors with five HC-Y188V subunits remain electrically silent because all five ACh binding sites are disabled, while receptors with four HC-Y188V subunits and one LC subunit exhibit the maximum observed current amplitude, indicating these receptors have one ACh binding site. Exemplar traces of channel openings from receptors with four HC-Y188V and one LC subunit, distinguished by current amplitude, show that in the absence of calcium channel openings are brief and occur in isolation, whereas in their presence channel openings are prolonged and occur in clusters (Fig. 4a, b), mirroring the observations from concatemeric receptors. A plot of current amplitude of each cluster against its duration exhibits five distinct horizontal stripes of points corresponding to receptors with zero to four HC-Y188V subunits. Notably, a sixth stripe of points corresponding to receptors with five HC-Y188V subunits is absent, confirming this stoichiometry, if present, is electrically silent. Moreover, without calcium, receptors with four HC-Y188V and one LC subunit show a compressed stripe of points spanning only brief durations (Fig. 4a), whereas with calcium, these receptors show an extended stripe spanning durations from brief to long (Fig. 4b). Receptors with three or fewer HC-Y188V subunits show stripes spanning durations from brief to long regardless of the presence of calcium (Fig. 4a, b). Bar graphs of the mean duration of all clusters affirm that receptors with one agonist binding site show brief unpotentiated channel openings without calcium, while all other combinations of binding site number and the presence or absence of calcium show long clusters of potentiated channel openings.
Fig. 4PNU-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
To further quantify potentiation, we plot the fraction of clusters with more than N channel re-openings, where N is an integer, against the number of times the channel re-opened per cluster. Here, a cluster with zero re-openings corresponds to a single channel opening flanked by long closings, while a cluster with one re-opening corresponds to two channel openings separated by a brief closure. A plot of the fraction of clusters with greater than N re-openings against N is expected to decay from one to zero, with a rapid decay indicating few re-openings per cluster and a slow decay indicating numerous re-openings per cluster. The number of exponential components in the decay indicates the minimum number of kinetically distinct closed states to which a cluster may transition, while the relative weight of each component indicates the probability of transition to a specific closed state. For receptors with one agonist binding site, without calcium, we observe a rapid decay in the number of re-openings per cluster, well fitted by a single brief exponential (Fig. 4a). Conversely, with calcium, the decay markedly slows, requiring a fit by the sum of two prolonged exponentials (Fig. 4b). By contrast, receptors with two binding sites exhibit a slow biexponential decay both without and with calcium (Fig. 4a, b). The mean number of re-openings per cluster for each exponential component, subunit stoichiometry and experimental condition is presented in Table 1. To summarize, our studies using both concatemeric receptors and electrical fingerprinting reveal that unlike receptors with multiple agonist binding sites, receptors with just one agonist binding site require calcium for PNU potentiation.
Table 1 Analysis of channel re-opening for receptors with reduced numbers of agonist binding sitesStructural basis of the effect of calcium on occupancy dependent PNU potentiationTo evaluate calcium binding sites linked to PNU potentiation, we took advantage of recent cryo-electron microscopic structures of the α7 nAChR. These structures revealed a ball-shaped density consistent with a calcium ion positioned between the extracellular and transmembrane domains of each subunit [14] (Fig. 1). Notably, an analogous density was absent in a subsequent α7 structure obtained without added calcium [16]. Surrounding this calcium ion density are four anionic residues, E45, D44, D42, and E173 originating from the extracellular domain (Fig. 5a). To determine the impact of these residues on PNU potentiation, we engineered mutations of each residue individually within the HC subunit. We then co-expressed each mutant subunit with the LC subunit and recorded single channel currents elicited by ACh together with PNU, either without or with calcium. We then determined the stoichiometric ratio of subunits based on current amplitude and analyzed mean cluster duration and channel re-opening for each amplitude class.
Fig. 5Contributions 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
Among the four mutants at the calcium site, only E45V had a discernable impact on mean cluster duration and channel re-opening. Receptors harboring five HC-E45V subunits (zero intact calcium sites) show brief channel openings flanked by long closings, regardless of the presence of calcium, indicating block of PNU potentiation (Fig. 5b). However, receptors with four HC-E45V subunits and one LC subunit show brief unpotentiated channel openings without calcium, but with calcium they show long clusters of potentiated openings, indicating the single copy of E45 within the LC subunit restores calcium-dependent PNU potentiation. In contrast, receptors with three or fewer HC-E45V subunits and two or more LC subunits show long potentiated clusters regardless of the presence of calcium, showing that two or more copies of E45 per pentamer enable calcium-independent PNU potentiation. Analyses of channel re-opening reveals changes parallel to those in mean cluster duration: a rapid, single exponential decay of channel re-opening corresponding to brief cluster duration, and a slow, biexponential decay corresponding to prolonged cluster duration (Table 2). Thus, in receptors with five agonist binding sites, E45 is pivotal in determining PNU potentiation either without or with calcium.
Table 2 Analysis of channel re-opening for receptors with reduced numbers of intact calcium binding sitesIn contrast to receptors harboring E45V, those containing D44N, regardless of the number of mutants per receptor, show long clusters of channel openings either without or with calcium (Fig. 5c; Table 2). Similarly, PNU potentiation is largely preserved in receptors with the mutants D42N and E173Q either without or with calcium (Fig. S3; Table 2). Thus, in receptors harboring five agonist binding sites, D42N, D44N and E173Q are dispensable for PNU potentiation, regardless of the presence of calcium.
Interdependence of agonist and calcium binding sites in PNU potentiationTo investigate the dynamic interplay among agonist binding sites, calcium binding sites, and PNU potentiation, we manipulated the number of agonist and calcium binding sites and recorded single channel currents from the resultant receptors. Inherent to this experiment, mutations of the agonist and calcium sites are located in different subunits so that any changes compared to the control subunit combination would manifest via inter-subunit interactions. We first recap the results for the control subunit combination HC-Y188V plus LC, which alters the number of agonist sites while preserving all five calcium sites (Fig. 4). Receptors with one agonist site and five intact calcium sites, denoted 1/5 in Fig. 6a, show brief mean cluster durations without calcium but long mean cluster durations with calcium. Analyses of channel re-opening confirms that brief clusters remain unpotentiated, exhibiting a single rapid exponential decay, while long clusters are potentiated, exhibiting a prolonged biexponential decay (Table 1).
Fig. 6Interdependence 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
For receptors with one agonist binding site, four intact calcium sites, and one calcium site containing any of the four mutants, denoted 1/4 in Fig. 6b–e, we observe profiles similar to those of the control receptor with one agonist site and five calcium sites: brief unpotentiated channel openings without calcium and long potentiated openings with calcium. Furthermore, analyses of channel re-opening reveals a rapid decay fitted by a predomintly single exponential without calcium, but a slow decay fitted by a prolonged single or double exponential with calcium (Fig. 6b–e; Table 3). Thus, for receptors with one agonist binding site, introducing a single copy of a mutation of any of the five residues at the calcium site has little effect either without or with calcium.
Table 3 Analysis of channel re-opening for receptors with reduced numbers of agonist and intact calcium binding sitesFor the control subunit combination HC-Y188V plus LC, receptors with two agonist sites and five intact calcium sites, denoted 2/5, long clusters of potentiated openings are observed either without or with calcium (Fig. 6a). Analyses of channel re-opening confirms that the long clusters consist of many openings in quick succession and that the decay is prolonged and biexponential (Fig. 6a, red decay curves; Table 1). In contrast, receptors with two agonist sites, three intact calcium sites, and two calcium sites with either E45V, D42N, or D44N mutations, denoted 2/3, show brief unpotentiated clusters without calcium (Fig. 6b–e; Table 3). However, these receptors show long clusters of potentiated channel openings with calcium. Notably, the analogous 2/3 receptors with the mutation E173Q differ from the others in showing long clusters of potentiated channel openings regardless of the presence of calcium. Thus, in receptors with two agonist sites, increasing the number of mutant calcium sites unmasks calcium-independent contributions of E45, D42, and D44 to PNU potentiation, and distinguishes these contributions from those by E173.
For the control subunit combination HC-Y188V plus LC, receptors with three agonist sites and five intact calcium sites, denoted 3/5, long clusters of potentiated channel openings are observed either without or with calcium (Fig. 6a). Conversely, receptors with three agonist sites, two intact calcium sites, and three calcium sites containing the E45V mutation, denoted 3/2, show brief clusters without calcium and prolonged clusters with calcium (Fig. 6b). Analyses of channel re-openings confirm that the brief clusters remain unpotentiated, primarily occurring as solitary brief openings, while the long clusters are potentiated, occurring as many openings in quick succession (Table 3). In contrast, the analogous 3/2 receptors with either D42N, D44N, or E173Q show long clusters either without or with calcium (Fig. 6c–e; Table 3). Thus, in receptors with three agonist sites, increasing the number of mutant calcium sites further differentiates among the residues at the calcium binding site: E45V abolishes calcium-independent PNU potentiation, whereas D42N, D44N, and E173Q have no discernable effect.
Lastly, for the control subunit combination HC-Y188V plus LC, receptors harboring four or five agonist sites and five intact calcium sites, denoted 4/5 or 5/5, respectively, long potentiated clusters of channel openings are observed either without or with calcium (Fig. 6a). In contrast, receptors containing four or five agonist sites, one or zero intact calcium sites, and four or five calcium sites with E45V, denoted 4/1 or 5/0, show brief unpotentiated openings either without or with calcium (Fig. 6b). Thus, increasing the number of agonist sites while increasing the number of mutant calcium sites reveals that at least two copies of E45 are required for calcium-dependent PNU potentiation. However, the analogous 4/1 and 5/0 receptors with either D42N, D44N, or E173Q mutations show long potentiated clusters of openings regardless of the presence of calcium. Furthermore, analysis of channel re-opening with calcium distinguishes among the D42N, D44N, and E173Q mutations, revealing a distinct signature of time constants and relative weights of the components of the biexponential decay for each mutant (Fig. 6c–e; Table 3). Our overall observations of the interplay between agonist and calcium binding sites suggests PNU potentiation relies on a delicate balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites.
Extracellular calcium binding sites do not impact PNU potentiationPreviously, we showed that calcium enhances α7 activity in the presence of low concentrations of ACh alone [19]. Calcium increased the frequency and mean duration of ACh elicited channel openings, effects that required a pair of anionic residues from the principal and complementary faces of each agonist binding site. Consequently, we asked whether this extracellular calcium binding site impacts the ability of calcium to modulate agonist occupancy dependent PNU potentiation. To investigate the possible impact of this extracellular calcium binding site, we co-expressed the HC subunit containing the mutation E185Q at the principal face with the wild-type LC subunit and analysed PNU potentiaton across all stoichiometric combinations of subunits. In contrast to our observations for mutations of E45, long clusters of channel openings are observed for all stoichiometric forms containing E185Q regardless of the presence of calcium (Fig. S4a). Next we co-expressed the HC-Y188V subunit with the LC-E185Q subunit and recorded single channel currents in the presence of ACh and PNU (Fig. S4b). Receptors with one agonist binding site show brief unpotentiated channel openings without calcium and long clusters of potentiated openings with calcium. In contrast, receptors with two or more agonist binding sites show long clusters of channel openings regardless of the presence of calcium. These observations with receptors comprised of the HC-Y188V plus LC-E185Q subunits mirror those for control receptors comprised of HC-Y188V plus LC subunits, indicating that although E185 is essential for potentiation of α7 activated by ACh alone, it does not impact the relationships between agonist binding sites, calcium binding sites, and PNU potentiation.
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