Abstract
The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, generated through alpaca immunization with cells expressing the extracellular domain of the human α7-nAChR, are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4β2 and α3β4. E3 acts as a slowly associating type I positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not significantly altering the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.
Competing Interest Statement
QL, AN, GA, PJC, PL, MP, NB are inventors of patent application US 63/383,099 that covers the VHH and therapeutic uses thereof.
List of Abbreviations
- 5-HT3A
- 5-hydroxy tryptamine type 3 subunit A
- α-Btx
- α-Bungarotoxin
- ACh
- acetylcholine
- Ago-PAM
- positive allosteric modulator with agonist properties
- BSA
- bovine serum albumin
- cDNA
- complementary deoxyribonucleic acid
- CDR
- complementarity-determining region
- cryo-EM
- cryogenic electron microscopy
- DMEM
- Dulbecco’s modified Eagle medium
- DMSO
- dimethyl-sulfoxide
- ECD
- extracellular domain
- EDTA
- ethylenediaminetetraacetic acid
- ELIC
- Erwinia ligand-gated ion channel
- ELISA
- enzyme-linked immunosorbent assay
- FBS
- fetal bovine serum
- GABA
- γ-aminobutyric acid
- GFP
- green fluorescent protein
- hα7-nAChR/m5-HT3A
- human α7-nAChR/mouse 5-HT3A
- HEK
- Human embryonic kidney
- HRP
- horse radish peroxidase
- ICD
- intracellular domain
- Ig
- immunoglobulin
- IRES
- internal ribosome entry site
- mRNA
- messenger ribonucleic acid
- nAChR
- nicotinic acetylcholine receptor
- NAM
- negative allosteric modulator
- OD
- optical density
- PAM
- positive allosteric modulator
- PBS
- phosphate buffered saline
- PBS-BSA
- PBS with 1% BSA
- PBST
- PBS with Tween 20
- PCR
- polymerase chain reaction
- PFA
- paraformaldehyde
- SAM
- silent allosteric modulator
- TCEP
- tris(2-carboxyethyl) phosphine
- TMD
- transmembrane domain
- VHH
- variable domain on heavy chain.