Abstract
M-currents are known to be inhibited by stimulating Gq-coupled receptors such as M1 receptor. M-currents can be reconstituted by expressing both KCNQ2/3 channels and M1 receptors either in mammalian cell lines or in Xenopus oocytes. However, the inhibition by stimulating co-expressing M1 receptors looks quite different in each expression system. Application of oxo-M or acetylcholine reduces KCNQ2/3 currents in HEK293T cells with no apparent change of voltage dependency while it shifts the conductance-voltage (G-V) curve of KCNQ2/3 in oocytes with no obvious reduction of maximum conductance. In this study, we aimed to know why there is a discrepancy between them. Among the signaling pathways of Gq, PIP2 hydrolysis and/or PKC activation are supposed to be important for the inhibition of M-current. When we applied 200 nM PMA (PKC activator) to KCNQ2 channels expressed in oocytes, we observed 20 mV positive shift of G-V curve with no significant reduction of maximum conductance. Next we incubated oocytes expressing KCNQ2 channels in 10 μM wortmannin for 30 min to reduce PIP2 and found that a minor shift of G-V curve was seen although only 20% of maximum currents were remained. These results suggest that PIP2 breakdown and PKC contribute to M-current inhibition differently: the maximum conductance is affected by PIP2 concentration and the voltage dependency is affected by phosphorylation by PKC. In HEK293T cells, V1/2 of the G-V curve was around -20 mV and close to V1/2 in PMA-treated oocytes. In addition, the currents were less sensitive to PMA. KCNQ2/3 channels may be constitutively phosphorylated in HEK293T cells. [Jpn J Physiol 55 Suppl:S127 (2005)]
