α7 nAChR activation by the Y674-R685 fragment from the SARS-CoV-2 S protein in the presence of potentiators.
Our previous MD simulations of the complex formed between the α7 nAChR and the Y674-R685 fragment from the SARS-CoV-2 S protein suggested the potential of the Y674-R685 region to interact with conserved aromatic residues within the binding pocket of the receptor (Fig. 1C) . To establish unequivocally the existence of molecular functional interactions between this region of SARS-CoV-2 S protein and the human α7 nAChR, we evaluated whether the synthetic fragment could elicit macroscopic and high-resolution single-channel currents.
The macroscopic responses of the human α7 nAChR expressed in Xenopus oocytes to the applications of the Y674-R685 fragment at a range of concentrations (1 pM-10 mM) were examined along with control ACh-evoked responses from the same cells (Fig 2A). As shown in the figure, Y674-R685 did not elicit detectable currents in contrast to the robust responses elicited by 100 µM ACh. After 5 min wash, receptors remained responsive to subsequent control applications of ACh (Fig. 2A).
Single-channel currents from cell-attached patches from BOSC-23 cells expressing human α7 nAChR were also recorded, thus allowing for more detailed mechanistic information. Recordings were carried out in parallel with control experiments with ACh as the agonist to confirm the presence of functional α7 nAChRs in the same batch of cells. ACh (10-100 µM) evoked isolated brief openings or less often short bursts composed of a few openings in quick succession, which correspond to activation of the same receptor molecule [18,21,22,24,25] (Fig. 2B). In contrast, channel activity was not detected at a range of Y674-R685 concentrations in a total of 21 patches from different cell transfections (1 pM, n=3; 1 nM, n=3; 1 µM, n=8; 10 µM, n=3; 100 µM, n=4) (Fig. 2B).
Given that α7 nAChR activation in the presence of ACh occurs with low open probability as very brief opening events (Fig. 2B), we sought to amplify this effect using a potentiator. PNU-120596, a type II positive allosteric modulator (PAM), has been extensively used as a tool in α7 functional studies due to its ability to increase the probability of agonist-elicited channel opening and open-channel durations and to reduce desensitization . We, therefore, examined whether Y674-R685 elicits α7 channel activity in the presence of PNU-120596. Note that by itself, PNU-120596 cannot induce channel activation .
The macroscopic currents elicited by 1 µM Y674-R685 in the presence of 10 µM PNU-120596 were recorded. Under these conditions, small currents in the low nA order were detected in 20% of the oocytes tested (n=25) whereas neither Y674-R685 nor PNU-120596 on their own elicited currents (Fig. 3A).
To gain more insights into how Y674-R685 activates the α7 nAChR in the presence of PNU-120596, we explored its effects at the single-channel level. ACh-elicited activity in the presence of 1 µM PNU-120596 is profoundly different to that in its absence (Fig. 3B). Instead of the brief isolated openings, channel activity shows long periods of high-frequency openings, named clusters, with a mean duration of about 1-3 s and an amplitude of 10 pA (-70 mV). A cluster corresponds to the activation episode of the same receptor that recovers from desensitization and oscillates between open and closed states before reaching again the more stable non-conducting desensitized state . Clusters are composed of bursts with mean durations of ~300-500 ms, which comprise successive openings separated by very brief closings (Figs. 3B and 4) [23,28].
In the presence of 1 mM PNU-120596, Y674-R685 was capable of eliciting channel activity at a wide range of concentrations (1 pM to 10 mM), indicating that this region of the S protein can activate α7 nAChRs (Fig. 3B). Since the frequency of channels is variable among patches due to variations in receptor expression levels, parallel control recordings in the presence of ACh were made. When ACh and PNU-120596 were co-applied, >98% of patches showed channel activity (active patches), and the long-duration clusters described above appeared at high frequency as reported before  (Fig. 3B). In the presence of PNU-120596 and Y674-R685, the percentage of active patches was lower than in the presence of ACh: 65% (n=23, N=4; 1 pM Y674-R685), 40% (n=15, N=4; 1 nM Y674-R685), 67% (n=15, N=4; 1 mM Y674-R685), and 62% (n=13, N=3; 10 mM Y674-R685). Also, channel activity evoked by Y674-R685 was much more infrequent and interspaced by long silent periods when compared to that evoked by ACh (Fig. 3B). It is important to note that this type of experiments does not allow for a precise comparison of channel frequency since this parameter may be affected by the variability in the number of receptors in each patch. Nevertheless, at 1 pM Y674-R685, the frequency of channel activation episodes was very low; albeit the active patches showed long clusters resembling those elicited by ACh and PNU-120596 (Figs. 3B and 4).
The increase in Y674-R685 concentration resulted in profound changes in the channel activity pattern, as clearly illustrated in the recordings shown in Fig. 3B. The frequency of opening events appeared to increase, but the duration of the openings and the activation episodes were reduced with increasing concentrations. The typical long-duration clusters were completely absent at Y674-R685 concentrations higher than 1 µM, at which activation occurred mainly as isolated openings or in short bursts (Fig. 3B and 4).
To define the properties of the activation episodes elicited by the Y674-R685 fragment at different concentrations, the mean durations of openings, bursts, and clusters in the presence of PNU-120596 were determined (Fig. 4). At 1 pM Y674-R685, the mean durations of the longest openings, bursts and clusters were 141 ± 59 ms, 417 ± 113 ms, and 2330 ± 673 ms, respectively (n=3). These values were similar to those determined in the presence of 10 µM ACh: 148 ± 12 ms for the slowest open component (p=0.859, n=3), 550 ± 38 ms for bursts (p=0.125, n=3), and 3048 ± 516 ms for clusters (p=0.217, n=3), and also comparable to those reported before for 100 µM ACh and 1 µM PNU-120596 [23,28]. Although the mean duration of clusters between 10 µM ACh and 1 pM Y674-R685 was similar, the relative area of the component corresponding to clusters in the histogram was smaller when Y674-R685 was the agonist (relative areas were for ACh= 0.44 ± 0.09 and for Y674-R685= 0.21 ± 0.08; p=0.0264) (Fig. 4). With the increase of Y674-R685 concentration, dwell time distributions for open, bursts and clusters were shifted to briefer durations. The slowest component of each histogram became progressively briefer with increasing Y674-R685 concentrations (Fig. 4). The mean durations were 48.2 ± 13.6 ms (1 µM, n=3) and 14.5 ± 4.3 ms (10 µM, n=4) for openings; 70.2 ± 19.7 ms (1 µM, n=3) and 20.7 ± 8.0 ms (10 µM, n=4) for bursts; and 104.2 ± 41.6 ms (1 µM, n=3) and 29.9 ± 14.8 ms (10 µM, n=4) for clusters. Mean values were statistically significantly different to those determined in the presence of 10 µM ACh and 1 µM PNU-120596 (p=0.000665 and p=0.00000431 for open; p=0.0000403 and p= 0.00000106 for burst; p=0.000597 and p=0.0000681 for cluster, for 1 and 10 µM Y674-R685, respectively). At high Y674-R685 concentrations (e.g., 10 µM), the mean open duration was similar to the mean burst and cluster durations (Fig. 4), indicating that openings occurred mostly in isolation and confirming the lack of the typical long-duration clusters corresponding to potentiated α7 responses.
To further confirm that channel activation can be elicited by Y674-R685 in the presence of PNU-120596 but not in its absence, single-channel recordings in the presence of the Y674-R685 fragment alone in the pipette solution (1-10 µM) were made. Again, no channel activity was detected in all patches. However, the addition of 1 µM PNU-120596 to the dish during the course of the recording resulted in the appearance of single-channel activity in most of the silent patches (Fig. 5). Again, the frequency of opening events was systematically markedly lower, and the durations were briefer than in the conditions in which ACh was the agonist (Fig. 5).
Together, these results confirm that Y674-R685 functionally interacts with the α7 nAChR. They also show that Y674-R685 acts as a very low-efficacy agonist since channel opening requires the presence of PNU-120596 and occurs with low probability, and that the increase in concentration is accompanied by a decrease in the duration of open channel lifetime and of the activation episodes.
Because PNU-120596 is a highly efficacious type II PAM with the capability of recovering receptors from desensitization, we also tested if Y674-R685 elicited channel activity in the presence of 5-hydroxyindole (5-HI), a type I PAM which induces clear potentiation but does not recover receptors from desensitization [21,23,30]. In the presence of 2 mM 5-HI, 100 µM ACh induced prolonged openings and bursts composed of successive openings which lasted about 4 ms (Fig. 6). The histograms showed that the duration of the slowest open component was ~4-fold longer (1.28 ± 0.35 ms, n=37, versus 0.30 ± 0.06 ms, n=38) and the mean burst duration was 8-fold longer (3.60 ± 1.29 ms, n=37, versus 0.46 ± 0.12 ms, n=38) than in the absence of the PAM. Replacing ACh by Y674-R685 (1 pM-10 µM) revealed α7 channel activity. However, the frequency of opening events was lower when compared to that elicited by ACh; only few events were detected during a 15-min recording period (Fig. 6). At 10 µM Y674-R685, the mean open duration was 0.73 ± 0.07 ms (n=3) and the mean burst duration was 0.85 ± 0.13 ms (n=3); both parameters were statistically significantly briefer than the corresponding mean durations in the presence of ACh and 2 mM 5-HI (p=0.0114 and 0.005 for mean open and mean burst durations, respectively). Also, the observation that in the presence of 10 µM Y674-R685 and 2 mM 5-HI the mean duration of openings was similar to that of bursts indicates that channel opening occurred mainly as isolated events instead of in quick succession forming activation episodes, as described before for recordings with the type II PAM PNU-120596.
Inhibition of α7 activity by Y674-R685 from S protein
To understand the molecular mechanisms driving the inhibitory effects of Y674-R685 evidenced by the dramatic decrease in open durations, we evaluated Y674-R685 effects on α7 activated by 10 µM ACh (Fig. 7A). Given the very brief duration of α7 channels, which is close to the time resolution of our system, we included PNU-120596 to better quantify a decrease in open durations. Whereas in the presence of 1 µM PNU-120596, 10 µM ACh led to an activation pattern composed of long clusters as described above, the inclusion of 10 µM Y674-R685 produced marked changes in this pattern: the long clusters were absent, and only markedly briefer bursts were detected (Fig. 7A). The mean duration of the slowest open component was 9.9 ± 2.6 ms (n=4), indicating a 15-fold decreased duration than in the absence of Y674-R685 (p=0.00000277). Also, the open duration was not different from the mean burst duration (11.2 ± 3.0 ms, n=4), indicating that most long openings occurred in isolation. Although clusters were not visually detected, we constructed cluster duration histograms using a critical time for cluster resolution between 10 and 20 ms, which is about 20 to 40-fold times longer than that used for burst-duration histograms. The mean duration of the slowest component of the cluster histogram was 14.1 ± 4.4 ms (n=4, p=0.0000663 with respect to clusters of 10 µM ACh and 1 µM PNU-120596), which is not different to that of bursts, thus confirming the lack of the long-duration activation episodes occurring in potentiated α7 channels.
The inhibitory effect was dependent on concentration since at 1 µM Y674-R685 the decrease in all parameters was smaller than at 10 µM, but still statistically significantly different from the control. At 1 µM Y674-R685, the mean durations were 45.7 ± 11.2 ms for open, 62.9 ±13.2 ms for bursts, and 90.5 ± 23.5 ms for clusters (n=3, p=0.000415, 0.0000296 and 0.000581 for open, bursts and clusters, respectively, with respect to ACh). The mean durations at 1 µM were also statistically significantly different to those at 10 µM Y674-R685 (p=0.00143 for mean open time, p=0.00056 for mean burst duration, and p=0.00122 for mean cluster duration), thus confirming the concentration-dependent inhibitory effect of Y674-R685.
To gain further insight into the mechanism driving the inhibitory effects of Y674-R685, we explored if channel activity elicited by ACh and PNU-120596 was affected by Y674-R685 as by methyllycaconitine (MLA), a reversible competitive α7 antagonist . To better assess the impact of MLA, we followed the strategy of filling the tip of the pipette with the buffer solution containing 10 µM ACh and 1 µM PNU-120596 and the tap with the same solution but including MLA (100 nM). This strategy allowed following in real-time the effects of the antagonist during the recording of ACh-activated channels. While at the beginning of the recording, the typical pattern of high channel activity and long clusters was observed, channel activity decreased over time, ultimately disappearing after about 10-15 min in the presence of MLA (Supplementary Fig. 2, n=3). Although the frequency of channel activity was markedly reduced, the kinetic properties of the remaining activation episodes were identical to those of the control. No reduction in the duration of clusters or bursts, as described in the presence of Y674-R685, was detected in the presence of MLA (Supplementary Fig. 2).
The results together suggest that the Y674-R685 region of the S protein acts as a non-competitive antagonist of α7. To further confirm this result, we determined the effects of Y674-R685 on the peak current responses evoked by an approximate EC50 concentration of ACh (100 mM). Application of Y674-R685 with ACh inhibited peak currents, with an IC50 of 1.8 ± 0.8 mM (n = 10), but inhibition was not complete (Fig. 7B). We then investigated whether the observed antagonism was competitive or not. For these studies, 1 μM Y674-R685 was co-applied with increasing concentrations of ACh (0.1- 2000 μM) (Fig. 7B). Compared to ACh alone, Y674-R685 co-application affected ACh efficacy, reducing the maximal currents elicited by ACh (Imax) by 30 ± 4% (n = 6) and slightly affecting its potency (EC50 = 80 ± 6 μM and 131 ± 92 μM) (n = 6). These results confirm the non-competitive antagonism of ACh responses by Y674-R685.