The C Terminus of the Human Nicotinic α4β2 Receptor Forms a Binding Site Required for Potentiation by an Estrogenic Steroid

Concentration–response data for ACh and βE2.A, The relative response to various concentrations of ACh, normalized to the response to 1 mm, in the absence of βE2. Human α4β2, open circles; rat α4β2,open triangles. Coapplication of 15 μmβE2 enhances the response from the human receptor (filled circles). Lines simply connect the points, whereas points show mean ± 1 SEM for three to six oocytes (derived from two batches).B, Potentiation of the human wild-type receptor response produced by coapplication of 15 μm βE2 plus various concentrations of ACh indicates that potentiation is greater at lower concentrations of ACh. The dotted lines show values of 1 (no potentiation), half the observed maximal potentiation, and maximal observed potentiation. Filled circles show data derived from oocytes tested at multiple concentrations (3 oocytes at each point), whereas open circles show pooled data from all oocytes tested (36 oocytes at 100 μm ACh and 84 oocytes at 1 μm ACh). C, The potentiation produced by various concentrations of βE2 coapplied with 1 μmACh indicate an EC₅₀ value of ∼10 μm for βE2. The apparent decrease between 20 μm and 30 μm βE2 is not statistically significant, although it could reflect mixed potentiation and block by βE2 at high concentrations. Filled circles show data derived from oocytes tested at multiple concentrations (3 oocytes at 3 μm, 2 oocytes at 20 μm, and 6 oocytes at other concentrations), whereas open circles show pooled data from all oocytes tested (13 oocytes at 3 μm, 84 oocytes at 15 μm, and 4 oocytes at 20 μm). The two symbols overlap at 3 and 15 μm βE2.

The onset and offset of potentiation are rapid. The response of a single oocyte to a long application of 1 μm ACh shows a slow decline reflecting desensitization (light trace). When a long application is made but the solution is switched from 1 μm ACh to 1 μmACh plus 15 μm βE2 during the application, the response is rapidly potentiated, and on return to 1 μm ACh alone rapidly returns to the same level as the response to 1 μmACh alone. In contrast, when the solution is switched to one containing 1 μm ACh plus 15 μm PROG, the response is rapidly blocked but recovers extremely slowly on return to 1 μm ACh alone. The responses were taken sequentially, with ACh alone first, followed by ACh plus βE2 and then ACh plus PROG. The holding current has been subtracted but the traces have not been normalized. There apparently was a small increase in response amplitude between the initial control application and the test applications.Bars indicate onset and duration of drug application.

Steroids tested for their ability to potentiate the human α4β2 nicotinic receptor. The structures of the steroids are identified by the abbreviations in the text, and values indicate the ratio of the response to coapplication of 1 μm ACh and 15 μm of the test compound to the response to 1 μm ACh alone in the same oocyte (±1 SD, with the number of oocytes tested in parentheses). The stereochemistry at all optically active centers is shown for βE2 andent-βE2; only partial stereochemistry for the other steroids is shown, but they are identical to that for βE2 at the additional centers. A, The structure in the top left corner shows the steroid ring backbone with the rings labeled A–D. The A ring is inbold, and arrows point to carbons 3 and 17 to indicate the importance of these positions. Although βE2 potentiates responses, its mirror image (ent-βE2) does not. B, Steroids containing a saturated A ring (3β17βED) or lacking a hydroxyl group at carbon 3 (3MeβE2 andmestranol) do not potentiate. C, Absence of a hydroxyl group at carbon 17 (E0,E1) or addition of a hydroxyl group at carbon 16 (E3) reduces potentiation. D, Potentiation occurs with an α-hydroxyl at carbon 17 (αE2), additional unsaturation in the B ring (Δ7 βE2), or an ethynyl group added to carbon 17 (ethynyl βE2).

Mutations in the C-terminal tail of the α4 subunit affect potentiation by βE2. Oocytes were injected with an α4 construct and wild-type β2 subunit. The first column shows the C-terminal amino acid sequence for each construct with the first entry (r, rat;h, human) indicating the origin of the sequence before the C terminus of the subunit. The sequence follows the predicted end of the M4 membrane spanning region, with the proline pair (PP) aligned vertically. The critical four residues are shown in bold, and the locations of mutations areboxed. The next column briefly describes each construct, followed by the n value. The bar graph to the right shows the mean ratio of the response to 1 μm ACh plus 15 μm βE2 to the response to 1 μm ACh alone in the same oocyte. The vertical lines at 1 and 3.85 indicate the average response from rat (no potentiation) and human α4β2 receptors. Error bars indicate the SEM. *indicates a p < 0.05 that the difference between the ratio for each construct and the ratio for human wild-type receptor, obtained from the same batch of oocytes (see Materials and Methods), arises by chance (evaluated using the t test assuming unequal variances); (*) is the same as *, except data from these three constructs were compared with the pooled data from all human wild-type receptors because wild-type responses were not determined from the same batch of oocytes.