Mutant human β4 subunit identified in amyotrophic lateral sclerosis patients impairs nicotinic receptor function

Abstract

Recently identified mutations in the genes encoding the neuronal nicotinic ACh receptor (nAChR) subunits in patients affected by sporadic amyotrophic lateral sclerosis (sALS) may represent a factor which enhances disease susceptibility, in particular in association with ambient causes such as cigarette smoking. In this work, we characterize the functional properties of nAChRs containing the β4R349C subunit, the mutation most frequently encountered in sALS patients. The mutation was coexpressed with wild-type α3 or α4 subunits or with mutant α4R487Q subunit, which has been detected in one patient together with β4R349C mutation. None of the functional parameters examined showed differences between α4β4 and α4R487Qβ4 nAChRs. By contrast, β4R349C mutation, independent of the companion α subunit, caused the reduction in potency of both ACh and nicotine, decreased the density of whole-cell current evoked by maximal transmitter concentrations, and altered the kinetics of ACh-evoked whole-cell currents. These data confirm that sALS-associated mutations in nicotinic subunits may markedly perturb cholinergic transmission in individuals bearing the mutations.

Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

“…We observed that two variants (red bars, Figure 1) significantly differed from α3β4 control currents. These were the β4 R348C variant (corresponding to the human R349C) which showed significantly reduced currents (0.12 ± 0.43 normalized amplitude increase) in agreement with previous reports (Moriconi et al, 2011; Richards et al, 2011), and the D447Y variant (corresponding to the human D444Y) that showed significantly larger currents (3.13 ± 0.24 normalized amplitude increase). This rare SNP reported by Weiss and colleagues (Weiss et al, 2008) has not been previously characterized functionally.…”

“…Quantitation of average current amplitudes revealed that the β4 point mutants A90I, T374I, and D447Y exhibited significantly larger responses to nicotine and that the R348C variant led to reduced nicotine-evoked current amplitudes, when compared to native α3β4 responses (Figure 4B). These results confirmed our electrophysiological studies in oocytes (Figure 1) and whole-cell recordings in transfected cells (Moriconi et al, 2011; Haller et al, 2012). …”

“…Representative traces of these recordings are shown in Figure 2A. It is interesting to note that the β4 R348C mutant not only leads to reduced agonist-induced currents, but also has slower kinetics of decay during ligand application (Figure 2A), consistent with reported electrophysiological recordings in cells (Moriconi et al, 2011). Upon overexpression, as previously observed for the native β4 (Frahm et al, 2011), nicotine-evoked currents increased 6.54 ± 0.43 fold for native β4, 6.12 ± 0.33 fold for β4A90I, 5.85 ± 0.68 fold for β4T374I, and 6.85 ± 0.07 fold for β4D447Y (Figure 2B).…”

“…Expression of these missense β4 variants in oocytes indicated that amino acid substitutions corresponding to 12 of these 14 SNPs led to changes in the electrophysiological properties of the channel (Liang et al, 2005; Haller et al, 2012). We further analyzed four of these SNPS in hippocampal neurons, and found that the two variants most associated with decreased risk of nicotine dependence, A90I and T374I (Haller et al, 2012), and a third variant D447Y, augmented nicotine-mediated α3β4 nAChR currents, while the fourth analyzed SNP, previously associated with sporadic amyotrophic lateral sclerosis (sALS) (Moriconi et al, 2011), reduced nicotine currents. Mice injected in the MHb with lentiviruses carrying the wild-type β4 subunit or β4 rare missense variants, showed aversion or preference to nicotine, depending on whether the variant increased or decreased nicotine currents.…”

Functional Characterization Improves Associations between Rare Non-Synonymous Variants in CHRNB4 and Smoking Behavior

“…The authors tested the T91I, R136W, S140G and M467V variants, and found relatively small changes in the EC 50 for ACh of a similar magnitude and direction to our observations. The second study examined the properties of the R349C variant, expressed in GH4C1 cells [33]. In this study, a relatively large increase in the EC 50 for ACh was found when the variant was expressed with the α3 subunit (3.2-fold) in comparison to our modest decrease (to 0.7-fold).…”