Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes.
It is well established that stimulation of G(q)-coupled receptors such as the M1 muscarinic acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that this muscarinic inhibition is mainly caused by the breakdown of PIP(2), the role of the subsequent activation of protein kinase C (PKC) is not well understood. By reconstituting M currents in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 microm oxotremorine M (oxo-M) induces a positive shift (4-30 mV, depending on which KCNQ channels are expressed) in the conductance-voltage relationship (G-V) of KCNQ channels. When we applied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large positive shift (17.8 +/- 1.6 mV) in the G-V curve for KCNQ2, while chelerythrine, a PKC inhibitor, attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP(2) had little effect on the G-V curve for KCNQ2 channels; although pretreating cells with 10 mum wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G-V curve was shifted only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T cells, the G-V curve seemed already to be shifted in a positive direction, even before activation of PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipette shifted the G-V curve in a negative direction suggests KCNQ2/3 channels are constitutively phosphorylated in HEK 293T cells.
Nakajo
Koichi
K
Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan. knakajo@nips.ac.jp
Kubo
Yoshihiro
Y
D000818:Animals; D002478:Cells, Cultured; D015640:Ion Channel Gating / Q000502:physiology*; D051656:KCNQ Potassium Channels / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D014982:Xenopus laevis
D051656:KCNQ Potassium Channels; D043643:Receptor, Muscarinic M1; D011493:Protein Kinase C