Abstract
Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.
Highlights
§ Supported by the Research Initiative for Scientific EnhancementMinority Biomedical Research Support-National Institutes of Health program (Grant 5R25GM61151)
Patch clamp recordings from oocytes expressing the ␣42 nicotinic acetylcholine receptor (nAChR) have demonstrated that single channel conductance depends on the ␣: ratio of the mRNA injected into the oocyte [29]
The results presented here demonstrate that the functional state of the ␣42 nAChR in the surface membrane of Xenopus oocytes can be manipulated by injecting different subunit ratios of mRNAs encoding for these two nAChR subunits
Summary
In Vitro Synthesis of mRNA and Oocyte Microinjection—Subunit mRNA was synthesized in vitro from linearized pGEMHE plasmid templates of Rattus norvegicus cDNA coding for ␣4 and 2 nAChR subunits using the mMessage mMachine RNA transcription kit (Ambion, Austin, TX). mRNA mixtures of ␣4 and 2 subunits were prepared at 1 g:4 g, 2 g:3 g, and 4 g:1 g ␣: ratios. We obtained ACh and nicotine dose-response curves (0.1, 1, 3, 10, 30, 100, and 300 M) before and after prolonged nicotine incubation For this nicotine incubation, oocytes were placed in L-15 medium supplemented with nicotine at a final concentration of 1 M for 24 h. Following chronic exposure to nicotine, incubated and non-incubated oocytes were dehydrated with Me2SO:methanol solution mixture, rehydrated with MOR2 buffer without EGTA (115 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1 mM Na2HPO4, 0.2 mM CaCl2, and 5 mM MgCl2, pH 7.4), and fixed in 4% paraformaldehyde. Oocytes were incubated in goat serum blocking solution prior to antibody treatment They were incubated overnight with a monoclonal antibody against the extracellular domain of the rat ␣4 nAChR subunit (mAb299, purchased from Covance Co.) and washed with MOR2 solution. The total fluorescence intensity value was calculated from each optical slice from the control and nicotine-incubated oocyte
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