Modulation of Gain-of-function α6*-Nicotinic Acetylcholine Receptor by β3 Subunits

Bhagirathi Dash,Ronald J Lukas

doi:10.1074/jbc.m111.322610

Abstract

We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))β4*- or α6(N143D+M145V)(L9'S)β2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.

Highlights

Function of physiologically important ␣6␤3*-nicotinic receptor is differentially impacted by ␤3 subunits
We replicated those findings in the current work, and we found that oocytes coexpressing Nicotinic acetylcholine receptors (nAChR) h␣6L9ЈS and h␤4 subunit cRNAs have marginally increased, but more reproducible, responses to nicotine
Because mecamylamine coexposure more than blocks inward currents produced by nicotinic agonists, leading under those conditions to production of apparent outward current responses, and does so in a concentration-dependent manner, we again interpret these effects as showing the ability of mecamylamine to block spontaneous opening of ␣6L9ЈSh␤4-nAChR channels (Table 1)

Summary

Background

Function of physiologically important ␣6␤3*-nicotinic receptor (nAChR) is differentially impacted by ␤3 subunits. ␣6*-nAChR are functional when expressed as hybrids of mouse and human ␣6 and other subunits, and there is function of some complexes containing ␤3 subunits mutated at specific residues in their second transmembrane domains (leucine- or valine-to-serine mutations at 9Ј or 13Ј positions; L9ЈS or V13ЈS) to confer gain-of-function effects (4, 15, 21). We hypothesized that wild-type ␤3 subunits would have the same effects, dominant-negative or potentiating, depending on the subunit combination investigated, on gain-of-function ␣6(L9ЈS or V13ЈS)*-nAChR as they did on wildtype ␣6*-nAChR This would help us assess whether any reduction or abolishment of function is due to altered open channel probability (21) or due to reduced surface expression of nAChR because ␤3 subunit incorporation facilitates formation of dead end intermediates (23). Our results indicated that whenever nAChR ␤3 subunits are incorporated into (␣6 or h␣6(N143DϩM145V))(L9ЈS or V13ЈS)*-nAChR, function is potentiated (i.e. there is higher agonist potency and larger magnitude responses) irrespective of whether there are dominantnegative or potentiating effects of ␤3 subunits on wild-type ␣6*-nAChR

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION