Varenicline, a new nicotinic ligand based on the structure of cytisine, has recently been approved by the U.S. Food and Drug Administration for use as a smoking cessation aid. Varenicline has been shown to be a partial agonist of ␣4␤2 receptors, and in equilibrium binding assays, it is highly selective for the ␣4␤2 receptor. In this study, we have examined the functional activity of varenicline at a variety of rat neuronal nicotinic receptors expressed in Xenopus laevis oocytes and assayed under two-electrode voltage clamp. We also find that varenicline is a potent, partial agonist at ␣4␤2 receptors, with an EC 50 of 2.3 Ϯ 0.3 M and an efficacy (relative to acetylcholine) of 13.4 Ϯ 0.4%. Varenicline has lower potency and higher efficacy at ␣3␤4 receptors, with an EC 50 of 55 Ϯ 8 M and an efficacy of 75 Ϯ 6%. Varenicline also seems to be a weak partial agonist at ␣3␤2 and ␣6-containing receptors, with an efficacy Ͻ10%. It is remarkable that varenicline is a potent, full agonist at ␣7 receptors with an EC 50 of 18 Ϯ 6 M and an efficacy of 93 Ϯ 7% (relative to acetylcholine). Thus, whereas varenicline is a partial agonist at some heteromeric neuronal nicotinic receptors, it is a full agonist at the homomeric ␣7 receptor. Some combination of these actions may be involved in the mechanism of varenicline as a smoking cessation aid.

We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the ␣4␤2 and ␣4␤4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat ␣4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEAbiotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate.Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that ␣4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas ␣4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of ␣4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepinebinding site on GABA A receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA A receptors.

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated alpha4beta4 or alpha4beta2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 microM) activated alpha4beta4 receptors in outside-out patches were similar in the absence and the presence of 100 microM zinc (3.0 +/- 0.1 and 2.9 +/- 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined alpha4beta4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 +/- 0.002) was increased 4.5-fold by 100 microM zinc (0.050 +/- 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 +/- 38 ms in the absence of zinc to 830 +/- 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.