10 research outputs found
The fifth subunit of the (α4β2)2β2 nicotinic acetylcholine receptor modulates maximal ACh responses
BACKGROUND AND PURPOSE:
The fifth subunit in the (α4β2)2α4 nicotinic acetylcholine receptor (nAChR) plays a determining role in the pharmacology of this nAChR type. Here, we have examined the role of the fifth subunit in the ACh responses of the (α4β2)2β2 nAChR type.
EXPERIMENTAL APPROACH:
The role of the fifth subunit in receptor function was explored using two-electrode voltage-clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4β2)2β2 receptors.
KEY RESULTS:
Covalent modification of cysteine substituted fifth subunit with a thiol-reactive agent (MTS) caused irreversible inhibition of receptor function. ACh reduced the rate of MTS reaction but the competitive inhibitor dihydro-β-erythroidine had no effect. Alanine substitution of conserved residues that line the core of agonist sites on α4(+)/β2(-) interfaces did not impair receptor function. However, impairment of agonist binding to α4(+)/β2(-) agonist sites by mutagenesis modified the effect of ACh on the rate of MTS reaction. The extent of this effect was dependent on the position of the agonist site relative to the fifth subunit.
CONCLUSIONS AND IMPLICATIONS:
We conclude that the fifth subunit in (α4β2)2β2 receptor isoform modulates maximal ACh responses. This effect appears to be driven by a modulatory, and asymmetric, association with the α4(+)/β2(-) agonist sites
A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release
Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca(2+) increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology
The Role of the Fifth Subunit on Sensitivity of α4β2-Containing Nicotinic Acetylcholine Receptors to Allosteric Modulators
The Role of the Fifth Subunit on Sensitivity of α4β2-Containing Nicotinic Acetylcholine Receptors to Allosteric Modulators
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XVIIth Little Brain Big Brain: an extraordinary meeting in extraordinary times.
Little Brain Big Brain is a biannual meeting organized and attended by young investigators. Since its inception in 1989, this meeting has brought together promising junior researchers in neurogastroenterology. The XVIIth meeting featured the latest basic, clinical and industry research in the field in a special post-pandemic edition
Asymmetric Agonist Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A Key Determinant of Agonist Efficacy
Role of the cys loop and transmembrane domain in the allosteric modulation of α4β2 nicotinic acetylcholine receptors
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.Allosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors and the α4β2-selective positive modulators 17-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiatingβEST is in the C-terminal (post-M4) region of the α4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate wi