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.

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA A receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the ␣ subunits of the receptor, whereas GABA binds to pockets in the interfaces between ␤ and ␣ subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ␤-␣ pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ␤-␣ pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the ␣ subunit from the same or the other ␤-␣ pair. We conclude that steroids potentiate the opening of the GABA A receptor induced by either agonist binding site.

Physostigmine is a well known inhibitor of acetylcholinesterase, which can also activate, potentiate, and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and β2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing three copies of α4 and two of β2 was potentiated at low concentrations of acetylcholine chloride (ACh) and physostigmine, whereas the form containing two copies of α4 and three of β2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh binding domain, the E loop, is a key determinant. Transferring the E loop from the β2 subunit to the α4 subunit resulted in strong inhibition, whereas the reciprocal transfer reduced inhibition. To control the number and position of the incorporated chimeric subunits, we expressed chimeric constructs with subunit dimers. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh binding site, whereas the lack of interface specificity indicates that interaction with a particular ACh binding site is not the critical factor.

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