Calcineurin Suppression of Alpha7 nAChR Currents in Xenopus Oocytes

Abstract

As part of our study of mechanisms underlying upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) in brain, due to chronic nicotine exposure, we are characterizing signaling pathways that influence the magnitude of α7 nAChR currents in Xenopus oocytes. In previous studies of Kv1.1 channels in oocytes, we found that calcineurin (CaN) and protein tyrosine kinases (PTKs) mediated calcium-dependent suppression of Kv1.1 currents, through both endocytosis of channels as well as effects on channel gating. Thus, we hypothesized that these molecules would similarly regulate nAChR currents. We first determined that 100 uM bath-applied genistein (an inhibitor of many PTKs) enhanced murine peak α7 nAChR currents in oocytes, by a factor of 2-3, as reported by others (Cho et al., 2005, J. Neuroscience). We also measured an increase in membrane capacitance (Cm), as expected if genistein's potentiation of peak currents was due to increased addition of channels to the plasma membrane via exocytosis. Conversely, injection of vanadate ion (VO43- ) into oocytes expressing α7 nAChRs produced a dramatic reduction (∼90%) in peak currents, but no clear decrease in Cm. Exposure of oocytes expressing α7 nAChRs to 10 uM cyclosporine A (CsA), a potent and specific inhibitor of CaN, increased the magnitude of α7 currents by a factor of ∼2, and also increased Cm. The facilitatory effect of CsA upon α7 current was blocked by 100 uM H-7, a non-specific inhibitor of serine/threonine PKs. The combination of genistein and CsA produced a greater enhancement of the α7 current than either agent alone; their combined effect was approximately additive. Our results suggest that CaN activity may provide a readout of the intracellular calcium concentration, due to recent activity of α7 nAChRs, and thereby negatively regulate the number of α7 nAChRs expressed on the plasma membrane.