Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3β4α5 nicotinic acetylcholine receptors

Abstract

The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3β4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3β4* receptor did not affect total [125I]-epibatidine binding or surface expression of the receptor. However, addition of α5D398 into α3β4* receptor decreased the maximal response to agonist without significantly affecting EC50 in aequorin intracellular calcium assay. α3β4α5N398 nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3β4α5 nAChRs led to significantly greater intracellular calcium release from IP3 stores relative to α3β4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC50 for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3β4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of a5 into an α3β4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling.

A CHRNA5 Smoking Risk Variant Decreases the Aversive Effects of Nicotine in Humans

“…This hypothesis is supported by large GWAS meta-analyses and in vitro studies that have characterized the function of 398 D4N and behavioral studies of α5 in animal models (Bailey et al, 2010;Bierut et al, 2008;Fowler et al, 2011;Frahm et al, 2011;Jackson et al, 2010;Kuryatov et al, 2011;Saccone et al, 2010;Tammimaki et al, 2012;Tobacco and Genetics Consortium, 2010). The CHRNA5 risk allele, rs16969968*A (398N), encodes a protein that forms nACh receptors with lower activity in several in vitro paradigms (Bierut et al, 2008;Kuryatov et al, 2011).…”

Two rare variations, D478N and D478E, that occur at the same amino acid residue in nicotinic acetylcholine receptor (nAChR) α2 subunit influence nAChR function

“…In this region the 398 th residue of nAChR hα5 subunit (i.e., D398), equivalent to the 471 st residue of nAChR hα2 subunit (i.e., E471) (Fig. 1; arrow mark), has drawn great attention for its role in ND and other diseases (Frahm et al, 2011; George et al, 2012; Hong et al, 2010; Kuryatov et al, 2011; Li et al, 2011; Tammimaki et al, 2012; Thorgeirsson et al, 2008). The N398 residue (i.e., the mutant/risk allele) of nAChR α5 subunit is predicted to alter the structure of intracellular vestibule/cytoplasmic portals of α3β4α5-nAChR (Frahm et al, 2011).…”

“…The D398N variation in nAChR α5 subunit commonly associated with ND and other diseases is mapped to the cytoplasmic vestibular regions (i.e. MA stretch) and is shown to modulate function of nAChRs both in vitro and in vivo (Frahm et al, 2011; George et al, 2012; Kuryatov et al, 2011; Tammimaki et al, 2012). In these studies it is shown that D398N variation in nAChR α5 subunit decreases the agonist response of α4β2- or α3β4-nAChRs (George et al, 2012; Kuryatov et al, 2011) or doesn’t affect the pharmacology of α3β4-nAChR activation (Li et al, 2011).…”

Nicotinic Receptors in Addiction Pathways

“…In vitro transfection studies have shown that introduction of the a5 subunit reduces the maximal a3b4* nAChR response to agonist activation and shifts the downstream signaling pathways (Tammimäki et al, 2012). Introduction of the D398N a5 subunit variant, which is linked to increased risk for nicotine dependence, further decreases agonist response at the a3b4* nAChR (Frahm et al, 2011;Tammimäki et al, 2012).…”

“…In vitro transfection studies have shown that introduction of the a5 subunit reduces the maximal a3b4* nAChR response to agonist activation and shifts the downstream signaling pathways (Tammimäki et al, 2012). Introduction of the D398N a5 subunit variant, which is linked to increased risk for nicotine dependence, further decreases agonist response at the a3b4* nAChR (Frahm et al, 2011;Tammimäki et al, 2012). A transgenic mouse model, Tabac, in which b4 subunit overexpression enhances a3b4* nAChR levels, has been shown to increase aversion to nicotine, an effect that is reversed by lentiviral transfection of the D398N a5 subunit into the MHb (Frahm et al, 2011).…”