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

Bhagirathi Dash,Minoti Bhakta,Yongchang Chang,Ronald J Lukas

doi:10.1074/jbc.m111.263673

Bhagirathi Dash, Minoti Bhakta + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.263673

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Abstract

Despite the apparent function of naturally expressed mammalian α6*-nicotinic acetylcholine receptors (α6*-nAChR; where * indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type β3 subunits abolishes the small amount of function typically seen for all-human or all-mouse α6β4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of α6 and β4 subunits with mutant β3 subunits harboring valine-to-serine mutations at 9'- or 13'-positions. There is no function when α6 and β2 subunits are expressed alone or in the presence of wild-type or mutant β3 subunits. Interestingly, hybrid nAChR containing mouse α6 and human (h) β4 subunits have function potentiated rather than suppressed by coexpression with wild-type hβ3 subunits and potentiated further upon coexpression with hβ3(V9'S) subunits. Studies using nAChR chimeric mouse/human α6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the α6 subunit N-terminal domain. More specifically, nAChR hα6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR hβ3 subunits on hα6hβ4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR hα6 subunits are involved in the gain-of-function effects of nAChR hβ3(V9'S) subunits on α6β2*-nAChR function. These studies illuminate the structural bases for effects of β3 subunits on α6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of α6 subunits are involved.

Highlights

Effects of nicotinic acetylcholine receptor (nAChR) h␤3V9؅S subunits on ␣6␤2*-nAChR function
There are at least six different nicotinic acetylcholine receptor3 ␣ subunits (␣2–␣7) and three nAChR ␤ subunits (␤2–␤4) expressed in the mammalian central nervous system (1). nAChR ␣7 subunits are thought principally to form homopentameric receptors when expressed in heterologous expression systems, whereas the other indicated subunits are thought to assemble into heteropentameric structures containing various combinations of ␣ and ␤ subunits. nAChR ␤3 and ␣5 subunits are considered to be “wild cards,” as they do not form functional receptors when expressed alone or in binary complexes with any other single subunit
The small amplitudes of current in the few oocytes that yielded functional receptors when injected with cRNA encoding nAChR h␣6 and h␤4 subunits plus wild-type h␤3 subunits confounded detailed analyses, studies done comparing nicotine and ACh efficacies and apparent potencies done on the same day after injection of the same batch of oocytes indicated that these agonists were efficacious (p Ͼ 0.05) (Table 1) at h␣6h␤4h␤3V9ЈS-nAChR

Summary

EXPERIMENTAL PROCEDURES

Chemicals—All chemicals for electrophysiology were obtained from Sigma. Fresh agonist (acetylcholine or nicotine) and antagonist (atropine or mecamylamine) stock solutions were made daily in Ringer’s solution and diluted as needed. Wild Type, Chimeric, or Point Mutation nAChR Subunits— cDNAs corresponding to human nAChR ␣6 (h␣6), ␤2 (h␤2), ␤3 (h␤3), or ␤4 (h␤4) subunits were excised from vectors containing them and subcloned into the oocyte expression vector pGEMHE. Data Analyses—Raw data were collected and processed in part using pClamp 10.2 (Molecular Devices, Sunnyvale, CA) and a spreadsheet (Excel; Microsoft, Bellevue, WA), using peak current amplitudes as measures of functional nAChR expression and results pooled across experiments (mean Ϯ S.E. for data from at least three oocytes). We made no attempt to measure or control for subunit combination-specific effects, but whenever preliminary studies revealed possible differences in peak current amplitudes, the findings were further confirmed across different subunit combinations using the same batch of oocytes and the same time between cRNA injection and recording. Whenever we make statements about results comparing ligand potencies and efficacies across subunit combinations, the observations are clear and significant (one-way analyses of variance followed by Tukey’s multiple comparison tests)

RESULTS

Imax concentration

DISCUSSION