Date Published: March 7, 2019
Publisher: Public Library of Science
Author(s): Maegan M. Weltzin, Andrew A. George, Ronald J. Lukas, Paul Whiteaker, Henning Ulrich.
Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.
Nicotinic acetylcholine receptors (nAChR) are members of the ligand-gated ion channel superfamily of neurotransmitter receptors, with the first-to-be-identified muscle-type nAChR serving as a prototype . In mammals functional pentameric nAChR subtypes with diverse pharmacological and biophysical properties, and distributions, are assembled from different combinations of nAChR subunits (α1—α7, α9, α10, β1—β4, γ, δ, ε). The endogenous neurotransmitter acetylcholine (ACh) activates function of all nAChR subtypes, and nicotine functions as an agonist of nAChR subtypes except those containing α9 subunits .
Data collected from nAChR expressed using unlinked subunits are depicted in gray (HS (α4β2)2β2-nAChR) or green (LS (α4β2)2α4-nAChR). Those collected from nAChR expressed using concatenated subunits are shown in magenta (HSP isoform) or cyan (LSP isoform). Primary data collected from concatenated LSP α4β2-nAChR containing mutant subunits are illustrated in red.
Previous single-channel studies of α4β2-nAChR function have usually examined populations with mixed stoichiometry. These have reported the existence of two, or occasionally three, distinct conductance states [23, 46–53]. These findings are broadly consistent with our own, which show a single conductance state associated with the HS α42-nAChR isoform, and two conductance states associated with the LS α4β2-nAChR isoform (one similar but not identical to that of the HS isoform, and one larger). The specific conductance values measured in this study also fall within the range of values reported previously, using nAChR subunits from a range of species, expression backgrounds, recording conditions (such as ionic strength and calcium concentrations), and patch configurations (see preceding citations). Importantly, the lower conductance associated with small amplitude LS α4β2-nAChR isoform (expressed using unlinked or linked subunits) openings was significantly different from the single conductance state measured for HS α4β2-nAChR (expressed using unlinked or linked subunits), proving that its appearance was not simply due to isoform contamination. Equally importantly, bursts containing multiple open amplitudes were extremely rare (< 0.05% of all events analyzed) and, in any case, were excluded from the analysis. As a result, the higher conductance state uniquely associated with LS (α4β2)2α4-nAChR cannot be an artifact produced by multiple, smaller-conductance, channel openings occurring simultaneously. Further evidence that small and large amplitude openings of the LS isoform correspond to distinct states of the receptor is provided by the fact that each is associated with a different, defined, open duration (with the large-conductance openings having longer open times for both ACh concentrations tested). Source: http://doi.org/10.1371/journal.pone.0213143