The rs16969968 variant in CHRNA5 is non-synonymous, resulting in an amino acid change (aspartate to asparagine) in the resultant α₅ nicotinic receptor subunit protein. Research has shown that this variant is functional. In vitro studies have demonstrated that nicotinic receptor complexes containing the α₅ receptor subunit featuring the aspartic acid variant exhibit a twofold greater maximal response to a nicotine agonist compared to α₅ receptor complexes containing the asparagine variant (i.e., the risk variant robustly associated with heavier smoking) (Bierut et al. 2008).

‘Knockout’ mouse models have enabled us to further explore the impact of specific genes on specific phenotypes. A knockout mouse is simply a mouse in which a specific target gene has been selectively disabled. Studying the physiological characteristics or behaviours of such mice (e.g., assessing their response to nicotine), provides insight into the mechanisms which link the knocked out gene to disease. The development of α₅ knockout mouse models has illustrated the role that the CHRNA5 gene plays in determining response to nicotine. Such mice are considered a model of individuals with reduced CHRNA5 gene function (i.e., carriers of the rs16969968 minor allele, associated with increased heaviness of smoking). Fowler et al. (2011) conducted an elegant series of experiments using the α₅ knockout mouse model. Based on a self-administration paradigm, they observed that knockout mice responded far more vigorously than wild-type mice (i.e., mice with no inactivated genes) for nicotine infusions at high doses (see Fig. 2a). Whilst wild-type mice self-titrated delivery of nicotine dose to achieve a consistent, desired level during each test session (~1.5 mg kg^-1), knockout mice did not, consuming greater amounts as dosage increased (see Fig. 2b). The authors proposed that deficient α₅ signalling attenuates the negative effects of nicotine that normally serve to limit its intake, a conclusion which fits well with human research (i.e., smokers carrying the rs16969968 risk allele are likely to smoke more heavily than their counterparts without the risk allele). Fowler and colleagues further demonstrated that this effect could be ‘rescued’ in α₅ knockout mice through injection of a lentivirus vector into the medial habenula (MHb), an area where α₅ subunits are typically densely expressed, rescuing expression of α₅ subunits in this region. The MHb projects to the interpeduncular nucleus (IPN). Notably, the pathway between the MHb and the IPN has previously been shown to regulate avoidance of noxious substances such as quinine (Donovick et al. 1970). Fowler and colleagues further observed reduced activity in the IPN in response to nicotine in knockout animals. Additionally, disruption of IPN activity was found to increase nicotine self-administration. However, both knockout and wild-type mice were indistinguishable in terms of responding for low doses of nicotine (see Fig. 2), indicative of shared experience of the rewarding, stimulatory effects of nicotine at low doses in both groups. These observations are complemented by a previous study by Jackson (2010), which also illustrated the initially shared and later differential experience of reward in response to an increasing dose of nicotine between α₅ knockouts and wild-types, in this instance illustrated using a conditioned place preference task.