Identification of N-terminal Extracellular Domain Determinants in Nicotinic Acetylcholine Receptor (nAChR) α6 Subunits That Influence Effects of Wild-type or Mutant β3 Subunits on Function of α6β2*- or α6β4*-nAChR

Abstract

Background: ␣6␤3*-Nicotinic receptors (nAChRs) are physiologically important but difficult to express heterologously. Results: Influences of ␤3 subunits on ␣6␤3*-nAChR function are impacted by ␣6 subunit N-terminal domain loop E residues. Conclusion: There are unexpected roles for the complementary face of the nAChR ␣6 subunit in receptor function. Significance: Novel medicinals acting at new sites on ␣6␤3*-nAChRs could be useful antidepressants and/or smoking cessation aids.

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

“…In this respect, C6 F223L differs from the classical ␣6 NTD / ␣3 TMD/ICL chimera (C1), but analogously the ␣6 NTD /␣4 TMD/ICL chimera has been shown to form functional receptors with ␤4 but not with ␤2 (24), and co-expression of this chimera with ␤2␤3 yields functional receptors (27). Furthermore, a complex pattern of subunit compatibilities has been observed for hybrid nAChRs formed from human and murine ␣6, ␤2, ␤4, ␤3, and ␤3 V273S subunits (29). All these findings bear witness to the allosteric nature of the nAChR complex and illustrate one of the potential shortcomings of the surrogate ␣6 subunit: although the Leu 223 residue and the ␣3-ICL in C6 F223L appear to have overcome the inborn molecular impediments in ␣6 for assembly and expression of functional ␣6␤4* nAChRs, other or additional elements in the subunit may counteract efficient formation of functional ␣6␤2* receptors.…”

“…Because of the extremely high expression levels of heterologously expressed proteins in this system, oocytes injected with WT ␣6␤4 cRNA actually form functional receptors, albeit agonist-evoked currents recorded from these have been reported to be minute (21,23,29). Thus, in contrast to the black-and-white functional rescue of ␣6␤4 function observed for the C6 F223L chimera in the FMP assay, a comparison of the functionalities of WT ␣6␤4 and C6 F223L ␤4 nAChRs in oocytes had to be based on the sizes of the maximal current amplitudes evoked by ACh in oocytes injected with comparable amounts of cRNA encoding for the two receptors.…”

“…First, co-expression of chimeric ␣6/␣3 or ␣6/␣4 subunits (␣6-NTD fused with ␣3-or ␣4-TMD/ICL) with ␤2, ␤2␤3, and ␤4 subunits results in formation of functional receptors in both mammalian cells and oocytes (22)(23)(24)(25)(26)(27). Second, the minute responses observed for ␣6␤2␤3 and ␣6␤4␤3 nAChRs in oocytes have been found to be dramatically enhanced by the introduction of a ␤3 V273S mutant in the receptors (28,29). Finally, expression of functional ␣6␤2␤3 and ␣6␣4␤2␤3 nAChRs in oocytes has recently been accomplished by linking subunits in pentameric constructs; this concatemerization somehow makes up for the absence of whatever cellular factors that enables the formation of functional wild type (WT) receptors in neurons (26).…”

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

“…As anticipated, only small currents were seen when attempts were made to express wild-type a6b4b3 receptors. Therefore, we introduced a gainof-function mutation in the b3 subunit, b3(V139S) (Dash et al, 2011), and this significantly improved expression levels. Once again, we will leave out the V139S notation for simplicity.…”

α-Conotoxin PeIA[S9H,V10A,E14N] Potently and Selectively Blocks α6β2β3 versus α6β4 Nicotinic Acetylcholine Receptors

“…We used ␣-Ctx PeIA as a template to create ligands with novel specificity and tested them on Xenopus oocytes expressing cloned nAChRs. We used rat and mouse ␣6/␣3 subunit chimeras to model the ␣6␤2 and ␣6␤4 ligand-binding domain because injection of nonchimeric ␣6 with ␤2 and ␤3 fails to reliably produce functional expression (Kuryatov et al, 2000;Dowell et al, 2003;Papke et al, 2005;Dash et al, 2011). However, comparison of data obtained from studies on heterologously expressed chimeric constructs of ␣6 with studies on native ␣6-containing nAChRs demonstrate that these chimeric constructs and native ␣6-containing nAChRs share a similar pharmacological profile (Bordia et al, 2007;Capelli et al, 2011;Pérez-Alvarez et al, 2012).…”