Functional Characterization of the α5(Asn398) Variant Associated with Risk for Nicotine Dependence in the α3β4α5 Nicotinic Receptor

Abstract

Smoking is a major cause for premature death. Work aimed at identifying genetic factors that contribute to nicotine addiction has revealed several single nucleotide polymorphisms (SNPs) that are linked to smoking-related behaviors such as nicotine dependence and level of smoking. One of these SNPs leads to an aspartic acid-to-asparagine substitution in the nicotinic receptor ␣5 subunit at amino acid position 398 [rs16969968; ␣5(Asn398)]. The ␣5 subunit is expressed both in the brain and in the periphery. In the brain, it associates with the ␣4 and ␤2 subunits to form ␣4␤2␣5 receptors. In the periphery, the ␣5 subunit combines with the ␣3 and ␤4 subunits to form the major ganglionic postsynaptic nicotinic receptor subtype. The ␣3␤4␣5 receptor regulates a variety of autonomic responses such as control of cardiac rate, blood pressure, and perfusion.In this paradigm, the ␣5(Asn398) variant may act by regulating autonomic responses that may affect nicotine intake by humans. Here, we have investigated the effect of the ␣5(Asn398) variant on the function of the ␣3␤4␣5 receptor. The wild-type or variant ␣5 subunits were coexpressed with the ␣3 and ␤4 subunits in human embryonic kidney 293 cells. The properties of the receptors were studied using whole-cell and singlechannel electrophysiology. The data indicate that the introduction of the ␣5(Asn398) mutation has little effect on the pharmacology of receptor activation, receptor desensitization, or single-channel properties. We propose that the effect of the ␣5(Asn398) variant on nicotine use is not mediated by an action on the physiological or pharmacological properties of the ␣3␤4␣5 subtype.

Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant

“…The previously described effects of ␣5 subunits on the efficiency of ␣3␤4* nAChR expression and possibly also on subunit associations/assembly could outweigh and obscure the effects of the ␣5(D398N) mutation. This could explain previous studies' conclusions that the effects of ␣5(Asp-389) incorporation were the same as those of ␣5(Asn-389) (18,25,37). However, using a pentameric concatemer approach, we were able to compare the function of uniform populations of (␣3␤4) 2 ␣5(Asp-389) versus (␣3␤4) 2 ␣5(Asn-389) nAChR.…”

“…Furthermore, ␣3, ␤4, and ␣5 nAChR subunits are commonly expressed in bronchial, epithelial, and lung cancer cells, where nAChR activation by nicotine has been proposed as a mechanism that may increase tumor initiation and/or growth (24). However, heterologous expression studies done to date have not identified functional differences induced by ␣5 variant incorporation into ␣3␤4* nAChR (18,25).…”

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

“…Removal of the functional block imposed by nicotine (probably acting as an open channel blocker), as a result of switching out to buffer application, leads to activation of the receptor by the residual nicotine that results in formation of a tail current. This is not unusual given that incorporation of accessory subunits into nAChR differentially affects various functional characteristics of the receptor (25).…”

Insights into the Neurobiology of the Nicotinic Cholinergic System and Nicotine Addiction from Mice Expressing Nicotinic Receptors Harboring Gain-of-Function Mutations

“…Although there are conflicting reports on ␣5 (Groot- Kormelink et al, 2001;Li et al, 2011), results from nearly all these studies are consistent: polar substitutions at the 9Ј or 13Ј position in the M2 domain ( Fig. 1) produce a marked (10-to 100-fold) leftward shift in the concentration-response curve, and thus create a useful feature to better understand these proteins in vitro and in vivo [in the useful "prime" nomenclature, the 1Ј position denotes the most N-terminal, cytoplasmic residue in the M2 region (Charnet et al, 1990)].…”

“…This observation continues to prove useful in interpretation of data derived from nAChR knock-in/ transgenic hypersensitive mice. Several studies on M2 domain mutations have since been conducted on many neuronal nAChR subunits, including ␣3 (Boorman et al, 2000), ␣4 (Fonck et al, 2005), ␣5 (Groot- Kormelink et al, 2001;Li et al, 2011), ␤3 (Boorman et al, 2000), and FIG. 1. nAChR M2 domain.…”