localization-plasma-membrane-intracellular-neuronal-nicotinic

Localization of Plasma Membrane and Intracellular Neuronal Nicotinic Acetylcholine Receptors Using Quantitative Imaging in Mammalian Cells

Quantification of neuronal nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane of mammalian cells is important for the development ...

University of Alaska Fairbanks

Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the HIV glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SARS-CoV-2 glycoprotein peptide [SCoV2P]), we identified that this area moderately inhibits α3β2, α3β4, and α4β2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system, and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293 cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.

SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in  oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

Cell membranes present barriers to the intracellular delivery of therapeutic agents. This impediment is frequently exacerbated by the hydrophobic characteristics of many such molecules, ultimately reducing the efficiency of their cellular uptake and therapeutic effectiveness. Therapeutics are being created that exploit natural bypass mechanisms by forming complexes with cell-penetrating peptides (CPPs) derived from viruses. However, current CPPs lack the ability to selectively target precise cellular macromolecules. As a result, they are distributed broadly and cause off-target side effects. Neurotropic CPPs derived from the rabies virus glycoprotein (RVG) can access the brain by binding to plasma membrane targets, including, but not exclusively, nicotinic acetylcholine receptors (nAChRs). To overcome this barrier of minimal target selectivity, we designed several chimeric peptides composed of regions from the RVG and -bungarotoxin, an α7 subtype-selective protein. Using human nAChRs expressed in  oocytes, we screened the selectivity of our peptides using two-electrode voltage clamp electrophysiology. We identified a peptide with improved α7 nAChR subtype selectivity and apparent potency compared to the control RVG peptide. Using mammalian Neuro-2a cells, we demonstrated that our peptide depends on α7 nAChR plasma membrane expression to internalize and carry small-molecule payloads into neuronal-like cells without significant cytotoxic effects. Our novel α7 nAChR subtype-selective CPP may be useful in research applications requiring cargo delivery. Translationally, our α7 nAChR-selective CPP holds potential to be a dual drug delivery system to transport cargo into the brain for the treatment of neurological diseases.

Article	Year
SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors. The Journal of biological chemistry\| PubMed ID: 37061001	2023
The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein. Frontiers in cellular and infection microbiology\| PubMed ID: 38836054	2024
Development of a novel alpha7-nicotinic acetylcholine receptor-selective cell-penetrating peptide for intracellular cargo transport. Drug delivery\| PubMed ID: 41319151	2025

Lahra Weber

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