Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.
Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC(inh)A01 and T16A(inh)A01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HE K 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC(inh)A01 and T16A(inh)A01 inhibited TMEM16A currents with IC50 values of 12, 58, 1.7 and 1.5 μM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC(inh)A01 and T16A(inh)A01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC(inh)A01 and T16A(inh)A01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.
ABSTRACT:The glycine receptor is an anion-permeable member of the Cys-loop ion channel receptor family. Synaptic glycine receptors predominantly comprise pentameric α1β subunit heteromers. To date, attempts to define the subunit stoichiometry and arrangement of these receptors have not yielded consistent results. Here we introduced FLAG and 6-His epitopes into α1 and β subunits, respectively, and imaged single antibody-bound α1β receptors using atomic force microscopy. This permitted us to infer the number and relative locations of the respective subunits in functional pentamers. Our results indicate an invariant stoichiometry of 2α1:3β with a subunit arrangement of β−α−β−α−β.Glycine receptor (GlyR) chloride channels mediate inhibitory transmission in the spinal cord and brain stem 1 . As they are considered potential therapeutic targets for indications including inflammatory pain, hyperekplexia, spasticity, tinnitus and breathing disorders [2][3][4][5] , there is widespread interest in understanding the molecular structure of their ligand binding sites for therapeutic development. GlyRs are members of the pentameric Cys-loop ion channel receptor family. Because ligandbinding sites are located at subunit interfaces, the pharmacological properties of each binding site are defined by the contributing subunits 6 . It is thus of interest to establish the subunits stoichiometry and arrangement in functional receptors to determine the number and type of subunit interfaces. Synaptic GlyRs are αβ heteromers, and receptors resulting from the cotransfection of varying amounts of α and β subunit RNA have uniform functional properties, implying a fixed stoichiometry 7,8 . Depending on their stoichiometry, heteromeric GlyRs may contain a mixture of α−α, α−β, β−α and β−β interfaces.The initial characterization of affinity-purified native GlyRs inferred that heteromeric GlyRs comprised 3α and 2β subunits 9,10 . This stoichiometry was supported by a subsequent functional analysis that showed the effect of a pore mutation was more dramatic when inserted into the α relative to the β subunit, implying an excess of α subunits per pentamer 11 . However, a study that compared the effects of mutations to corresponding glycine binding residues in α1 and β subunits concluded that β subunits predominated in α1β GlyRs 12 . They then showed that α1-β concatemers produced functional heteromers when co-expressed with β monomers but not when expressed alone or with α1 monomers. This was consistent with either a 2α:3β or a 1α:4β stoichiometry. Quantitation of radio-labeled methionine levels in recombinant α1 and α1β GlyRs allowed the authors to deduce a 2α1:3β stoichiometry, with an arrangement of β−α−β−α−β 12 .However, potential problems with using concatemers include dimer proteolysis 13 , dipentamer formation 14 and incomplete incorporation of fusion protein into individual pentamers 15 . These problems are more likely to occur when dimers are co-transfected with monomers than when fully concatenated pentamers are used 16,17 . Thus, as the ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.