Nicotine-Upregulation of Alpha 7 nAChR in Xenopus Oocytes

Abstract

The alpha 7 (α7) subtype of nAChR appears to play critical roles in learning, cognition, and various neuropathologies including nicotine addiction. Nicotine-upregulation of α7 Rs may play a significant role in the latter phenomenon. But whether nicotine-upregulation of α7 Rs reliably occurs, and its underlying mechanism(s), are largely unknown. Previous studies of α7 nAChRs heterologously expressed in Xenopus oocytes failed to observe nicotine-upregulation. These failures might have been due to intracellular accumulation of nicotine and its subsequent slow release from oocytes, resulting in desensitization of α7 Rs during functional assays. In our experiments, 12-14 hr exposure to nicotine (100 μM) 4-5 days post cRNA-injection (PI), followed by extensive washout, yielded statistically-significant ∼2-fold increases in peak α7 currents and net charge as determined by TEVC and α7-protein (by Western blot). Less-extensive washout, as well as 100 nM nicotine incubation, failed to produce upregulation. Instead, inactivated/desensitized currents were observed. GC/MS measurement of nicotine in the washout fluid confirmed that nicotine was continually released from oocytes. The concentration was ∼20 nM at 3-4 hr following washout onset, well in excess of the ∼ 3 nM IC50 value. Exposure of α7-expressing oocytes to cumulative washout fluid produced suppressed α7 currents (∼ 8% of controls). Similar to nicotine, methyllycaconitine, a cell-permeable competitive antagonist of α7 Rs, and carbachol, a stable membrane-impermeable agonist, also produced ∼ 2X-upregulation. However, ACh incubation (which can be hydrolyzed by the oocytes) failed to produce upregulation. We also found that chelation of intracellular Ca2+ by BAPTA-AM completely blocked nicotine upregulation. However, elimination of extracellular Ca2+ had no effect. These results suggest that persistent ligand-binding to α7 Rs (but not necessarily channel gating), leading to increases in [Ca2+]i, was critical for α7 R-upregulation. Several Ca2+-dependent signaling pathways were implicated in nicotine-upregulation of α7.