Carlson EC et al. (2023), The SARS-CoV-2 Virus and the Cholinergic System...

XB-ART-59556

Int J Mol Sci 2023 Mar 15;246:. doi: 10.3390/ijms24065597.

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The SARS-CoV-2 Virus and the Cholinergic System: Spike Protein Interaction with Human Nicotinic Acetylcholine Receptors and the Nicotinic Agonist Varenicline.

Carlson EC , Macsai M , Bertrand S , Bertrand D , Nau J .

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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. Although the pathophysiology of SARS-CoV-2 infection is still being elucidated, the nicotinic cholinergic system may play a role. To evaluate the interaction of the SARS-CoV-2 virus with human nicotinic acetylcholine receptors (nAChRs), we assessed the in vitro interaction of the spike protein of the SARS-CoV-2 virus with various subunits of nAChRs. Electrophysiology recordings were conducted at α4β2, α3β4, α3α5β4, α4α6β2, and α7 neuronal nAChRs expressed in Xenopus oocytes. In cells expressing the α4β2 or α4α6β2 nAChRs, exposure to the 1 µg/mL Spike-RBD protein caused a marked reduction of the current amplitude; effects at the α3α5β4 receptor were equivocal and effects at the α3β4 and α7 receptors were absent. Overall, the spike protein of the SARS-CoV-2 virus can interact with select nAChRs, namely the α4β2 and/or α4α6β2 subtypes, likely at an allosteric binding site. The nAChR agonist varenicline has the potential to interact with Spike-RBD and form a complex that may interfere with spike function, although this effect appears to have been lost with the omicron mutation. These results help understand nAChR's involvement with acute and long-term sequelae associated with COVID-19, especially within the central nervous system.

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Species referenced: Xenopus laevis
GO keywords: acetylcholine receptor activity

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	Figure 1. Effects of the spike protein containing the receptor-binding domain (Spike-RBD) protein at the concentration-response curve to acetylcholine (ACh) of the α4β2 nicotinic acetylcholine receptor. Typical currents were recorded in the same cell before (green traces) and during short (left-hand side) and long (right-hand side) exposure to 1 μg/mL of the Spike-RBD protein (red traces). Currents were elicited by brief exposure to ACh test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in seven cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 2. Effects of the spike protein containing the receptor-binding domain (Spike-RBD) protein at the concentration-response curve to acetylcholine (ACh) of the α4α6β2 nicotinic acetylcholine receptor complementary DNA. Typical currents were recorded in the same cell before (green traces) and during exposure to 1 μg/mL of the Spike-RBD protein (red traces). Currents were elicited by brief exposure to ACh test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in five cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 3. Effects of the spike protein containing the receptor-binding domain (Spike-RBD) protein at the concentration-response curve to acetylcholine (ACh) of the α3α5β4 nicotinic acetylcholine receptor. Typical currents were recorded in the same cell before (green traces) and during exposure to 1 μg/mL of the Spike-RBD protein (red traces). Currents were elicited by brief exposure to ACh test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in four cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 4. Effects of the SARS-CoV-2 spike protein S2 subunit at the human α4β2 nicotinic acetylcholine receptor. Typical currents were recorded in the same cell before (green traces) and during exposure to 1 μg/mL of the Spike protein S2 subunit (red traces). Currents were elicited by brief exposure to acetylcholine (ACh) test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in nine cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 5. Effects of SARS-CoV-2 Spike protein S1 subunit at the human α4β2 nicotinic acetylcholine receptor. Typical currents were recorded in the same cell before (green traces) and during exposure to 1 μg/mL of the Spike protein S1 subunit (red traces). Currents were elicited by brief exposure to acetylcholine (ACh) test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in six cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 6. Effects of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) B.1.1.529 spike protein containing the receptor-binding domain (Spike-RBD) protein at the human α4β2 nicotinic acetylcholine receptor. Typical currents were recorded in the same cell before (green traces) and during exposure to 10 μg/mL of the SARS-CoV-2 B.1.1.529 Spike-RBD protein (red traces). Currents were elicited by brief exposure to acetylcholine (ACh) test pulses with a series of concentrations. Currents were normalized to unity vs. the maximal value and average results obtained in five cells were plotted vs. the logarithm (log) of the ACh concentration. The green curve is the best fit obtained with the Hill equation. Areas under the curve are plotted on the lower right-hand side.
	Figure 7. Exposure to low, but sustained, applications of varenicline desensitize the human α4β2 nicotinic acetylcholine receptor. Typical currents evoked by brief acetylcholine test pulses (50 μM) were recorded in a cell expressing the complementary DNA encoding for the human α4β2 receptors were recorded first in control conditions (green trace) and then during exposure to a series of varenicline concentrations ranging from 0.03 to 300 nM.
	Figure 8. Peak inward currents evoked by 50-μM acetylcholine were recorded in a series of cells for control (n = 7; red) and during exposure to 1-μg/mL spike protein containing the receptor-binding domain (Spike-RBD) (n = 8; green) and normalized to unity vs. the response recorded in control. Continuous lines are the best fits obtained with the Hill equation. CTRL, control; log, logarithm.
	Figure 9. The area under the curve (charges) of the currents evoked by 50-μM acetylcholine recorded in a series of cells for control (n = 7; red) and during exposure to 1-μg/mL spike protein containing the receptor-binding domain (Spike-RBD) (n = 8; green) and normalized to unity vs. the response recorded in control. Continuous lines are the best fits obtained with the Hill equation. CTRL, control; log, logarithm.
	Figure 10. Peak inward currents evoked by 50-µM acetylcholine were recorded in a series of cells for control (n = 9; red) and during exposure to 10-µg/mL omicron S protein (n = 9; green) and normalized to unity vs. the response recorded in control. Continuous lines are the best fits obtained with the Hill equation. log, logarithm.
	Figure 11. (A) Experimental protocol to assay effects of spike protein containing the receptor-binding domain (Spike-RBD) with human nicotinic acetylcholine receptor (nAChR) subtypes. (B) Experimental protocol to assay effects of varenicline with Spike-RBD at human α4β2 nAChRs.

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