Subunit-dependent modulation of neuronal nicotinic receptors by zinc

Abstract

We examined the effect of zinc on rat neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes as simple heteromers of alpha2, alpha3, or alpha4 and beta2 or beta4. Coapplication of zinc with low concentrations of acetylcholine (</=EC(10)) resulted in differential effects depending on receptor subunit composition. The alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, and alpha4beta4 receptors exhibited biphasic modulation by zinc, with potentiation of the acetylcholine response occurring at 1-100 micrometer zinc and inhibition occurring at higher zinc concentrations. In contrast, alpha3beta2 receptors were only inhibited by zinc (IC(50) = 97 +/- 16 micrometer). The greatest potentiating effect of zinc was seen with alpha4beta4 receptors that were potentiated to 560 +/- 17% of the response to ACh alone, with an EC(50) of 22 +/- 4 micrometer zinc. Cadmium, but not nickel, was also able to potentiate alpha4beta4 receptors. Both zinc potentiation of alpha4beta4 receptors and zinc inhibition of alpha3beta2 receptors were voltage independent. The sensitivity of zinc potentiation of alpha4beta4 to diethylpyrocarbonate treatment and alterations in pH suggested the involvement of histidine residues. Zinc continued to inhibit alpha4beta4 and alpha3beta2 after diethylpyrocarbonate treatment. Application of a potentiating zinc concentration increased the response of alpha4beta2 and alpha4beta4 receptors to saturating ACh concentrations. The rate of Ach-induced desensitization of these receptors was unaffected by zinc. Our results reveal zinc potentiation as a new mode of neuronal nAChR modulation.

Fig. 1.

Zn²⁺ potentiates and inhibits neuronal nAChRs. A, Current responses of an α4β2-expressing oocyte to 10 μm ACh before, during, and after coapplication of 70 μm(top trace) or 1 mm Zn²⁺(bottom trace). Calibration: 60 nA, 20 sec.B, Current responses of an α3β2-expressing oocyte to 4 μm ACh before, during, and after coapplication of 70 μm Zn²⁺(top trace) or 1 mm Zn²⁺(bottom trace). Calibration: 300 nA, 20 sec. C, Current responses of an α4β4-expressing oocyte to 1 μm ACh before, during, and after coapplication of 100 μm Zn²⁺(top trace) or 3 mm Zn²⁺(bottom trace). Calibration: 200 nA, 20 sec.

Fig. 2.

The nature of neuronal nAChR modulation by Zn²⁺ is subunit dependent. The effects of Zn²⁺ coapplication on Ach-induced current responses are plotted as a percentage of the response to ACh alone (mean ± SEM of 3–6 oocytes). Some error bars are obscured by the symbols. The potentiating and inhibiting phases of the Zn²⁺effect were fit separately as described in Materials and Methods. Note the increase in axis range in C to accommodate the greater potentiation seen with α4β4 nAChRs.

Fig. 3.

Zn²⁺ preincubation eliminates potentiation transients seen with high Zn²⁺concentrations. A, Current responses of an α4β4-expressing oocyte preincubated with 0, 100 μm, and 1 mm Zn²⁺ for 20–30 sec before coapplication of 1 μm ACh. Calibration: 50 nA, 10 sec.B, Current responses during coapplication of various concentrations of Zn²⁺ and 1 μm ACh were plotted as a percentage of the response to 1 μm ACh alone recorded immediately before Zn²⁺ preincubation (mean ± SEM of 3 oocytes).

Fig. 4.

Cd²⁺ and Ni²⁺ are less effective than Zn²⁺at potentiating neuronal nAChRs. The effects of Cd²⁺(▿) and Ni²⁺ (⋄) on ACh (1 μm)-induced current responses of α4β4-expressing oocytes are plotted as a percentage of the current obtained with 1 μm ACh alone (mean ± SEM of 3–5 oocytes). Potentiating and inhibiting phases were fit separately as described in Materials and Methods. Data from Figure 2C showing the effect of Zn²⁺ (○) are included for comparison. Some error bars are obscured by symbols.

Fig. 5.

Diethylpyrocarbonate treatment abolishes Zn²⁺ potentiation but not Zn²⁺inhibition of neuronal nAChRs. A, The current response of an α4β4-expressing oocyte to 1 μm ACh before, during, and after coapplication of 100 μmZn²⁺ is shown on the left. After a 10 min treatment with 3 mm DEPC and a 10 min wash period, application of 100 μm Zn²⁺ to the same oocyte no longer potentiates the ACh response (right trace). Calibration: 250 nA, 20 sec. B, The effect of a 10 min incubation with various concentrations of DEPC on ACh (1 μm)-induced current responses in the presence of 100 μm Zn²⁺ (●) is plotted as a percentage of the response to ACh immediately before Zn²⁺ application. The effect of DEPC on current responses to 1 μm ACh alone (○) is plotted as a percentage of the response to ACh before DEPC treatment. Symbols and error bars represent the mean ± SEM of four sets of oocytes, each set consisting of three oocytes. Error bars are obscured by symbols.C, The effect of various concentrations of Zn²⁺ on the response of α4β4 receptors to 1 μm ACh before (♦) and after (⋄) treatment with 3 mm DEPC is plotted as a percentage of the response to ACh alone immediately before Zn²⁺ application (mean ± SEM of 3 oocytes). D, Block of α3β2-expressing oocytes by 1 mm Zn²⁺ was measured before (−) and after (+) treatment with 3 mm DEPC for 10 min and is plotted as a percentage of the response to 4 μm ACh immediately before Zn²⁺ application (mean ± SEM of 3 oocytes).

Fig. 6.

Zn²⁺ potentiation of α4β4 is sensitive to alterations in pH. Potentiation of the response to 1 μm ACh by 100 μm Zn²⁺ at pH 5.5, 7.2, and 8.0 is plotted as a percentage of the response to ACh alone (mean ± SEM of 3 oocytes; significant differences from pH 7.2: *p < 0.0001, **p < 0.005).

Fig. 7.

Zn²⁺ potentiation of α4β4 receptors and inhibition of α3β2 receptors are voltage independent.A, Current responses of an α4β4-expressing oocyte to 1 μm ACh before, during, and after coapplication of 50 μm Zn²⁺ at membrane holding potentials of −40 mV (left trace, calibration: 50 nA, 20 sec) and −90 mV (right trace, calibration: 200 nA, 20 sec). The traces were normalized for comparison. B, Current responses of α4β4-expressing oocytes to 1 μm ACh in the presence of 50 μm Zn²⁺ were recorded at various holding potentials and plotted as a percentage of the response to ACh alone (mean ± SEM of 3 oocytes). Current responses of α3β2 expressing oocytes to 4 μm ACh in the presence of 100 μm Zn²⁺ were recorded at various holding potentials and plotted as a percentage of the response to ACh alone (mean ± SEM of 3 oocytes).

Fig. 8.

Zn²⁺ increases the response of neuronal nAChRs to saturating ACh concentrations. ACh concentration–response relationships of α4β4- and α4β2-expressing oocytes in the absence (filled symbols) and presence (open symbols) of 50 μm Zn²⁺ are plotted as a percentage of the fit maximum response to ACh alone (mean ± SEM of 3 oocytes).A, Zn²⁺ (50 μm) significantly decreased the EC₅₀ of α4β4 receptors for ACh from 74 ± 22 to 23 ± 8 μm(p < 0.05) and significantly increased the maximal response to 160 ± 11% of the maximal response to ACh alone (p < 0.02). B, Zn²⁺ (50 μm) significantly increased the maximal response of α4β2 receptors to ACh to 140 ± 14% of the maximal response to ACh alone (p < 0.02).

Fig. 9.

Zn²⁺ modulation of receptors formed by chimeric α subunits. The effects of Zn²⁺coapplication on the Ach-induced current responses of α4–216-α3 β4 (●) and α3–216-α4 β4 (▴) receptors are plotted as a percentage of the response to ACh alone (mean ± SEM of 3 oocytes). Some error bars are obscured by symbols. The potentiation and inhibition curves for α4β4 and α3β4 taken from Figure 2 are shown for comparison (dashed lines).