A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-022-02947-8.


Supplementary Figures
Supplementary Figure 1. Overview of the three-dimensional structure of the S protein from SARS-CoV-2. The model shown here represents the complete SARS-CoV-2 S protein in the closed state after furin cleavage. This model was taken from (Casalino et al., 2020). The S1 and S2 subunit are coloured in green and orange, respectively. The glycans were omitted for simplicity. The Y674-R685 region (proposed to interact with nAChR) is shown with red spheres.
Activation of the human α7 nAChR by 1 pM Y674-R685 in the presence of the type I PAM, 5-HI.
Single-channel currents were recorded from cells expressing the human α7 nAChR in the presence of 2 mM 5-HI as a PAM and 1 pM Y674-R685. Membrane potential: -70 mV, Filter: 9 kHz. Burst duration histograms were constructed with data from 18 different patches. The resulting mean burst duration was similar to that of recordings in the presence of 5-HI and ACh as the agonist (Fig. 6).

Closed time histograms of channels activated by ACh and potentiated by PNU-120596 in the presence of different peptide concentrations.
Representative closed duration histograms of channels activated by 10 µM ACh and potentiated by 1 µM PNU-120596 in the absence or presence of different peptide concentrations (1 pM, 1 nM, 1 µM and 10 µM). The histograms were fitted by the sum of exponential functions by maximum likelihood using the program TACFit (Bruxton Corporation, Seattle, WA, USA). The duration and area of all components for each condition was measured and shown in the table.

Effect of the competitive antagonist MLA on single α7 channels activated by 10 µM ACh and potentiated by 1 µM PNU-120596 measured in real time.
To measure the effect of MLA on single channels of human α7 nAChR activated by 10 µM ACh and potentiated by 1 µM PNU-120596 in real time we used a patch clamp pipette in which the tip was filled with the buffer solution containing 10 µM ACh and 1 µM PNU-120596 and the shaft with the same solution but including MLA (100 nM). At the beginning of the recording, high channel activity was observed, which appeared as multiple levels of channels due to the opening of more than one channel at the same time (upper trace). As the recording progressed, channel activity decreased (middle trace), and the clusters showed the same duration as in the absence of MLA.
After about 10 min, channel activity was completely inhibited (lower trace). The traces correspond to different times of the same representative recording. Membrane potential: -70 mV, Filter: 3 kHz. (Oliveira et al., 2021). Examples of the most compact and extended conformations adopted Y674-R685 within the agonist binding site.

Examples from our previous MD simulations of conformations adopted by the Y674-R685 fragment when bound to the human α7 nAChR.
MD simulations of Y674-R685 bound to the human α7 nAChR show favourable binding (Oliveira et al., 2021).

Example of an open and semi-open conformation of loop C from our previous simulations
of the Y674-R685:α7 nAChR complex (Oliveira et al., 2021). The loop C is highlighted in red, and the principal and complementary subunits of the receptor are coloured in green and dark blue, respectively. The Y674-R685 fragment was omitted for simplicity. Single-channel currents elicited by 10 µM ACh and potentiated by 1 µM PNU-120596 were recorded in the absence (control) or presence of Y674-R685 at the indicated concentrations.

Supplementary
The open, burst and cluster durations were obtained from the corresponding histograms.
n indicates the number of recordings for each condition.
p corresponds to the resulting p value of the Student-t test in which each parameter was compared to that in the absence of Y674-R685.