Intracellular Zinc Potentiates KCNQ Channels through Modulation of their Sensitivity to PIP2

Abstract

KCNQ potassium channels which underlie neuronal M current play dominant roles in controlling neuronal excitability. Inhibition or genetic loss of M channels are implicated in many excitability disorders (e.g. epilepsy, pain, deafness, arrhythmia), while M channel openers retigabine and flupirtine are used clinically as anticonvulsant drugs and analgesics. Zinc pyrithione (ZnPy) is one of the recently discovered M channel openers which activity was previously attributed to the pyrithione moiety; ZnPy is also used as zinc ionophore. Here we show that the likely mechanism of ZnPy action on KCNQ channels is elevation of intracellular zinc which, in turn, activates the channels. Using patch-clamp recordings we show that 10μM ZnPy increased the recombinant KCNQ4 and KCNQ2/3 currents as well as native M current in DRG neurons by 2.4±0.4, 1.6±0.1 and 1.6±0.2 fold respectively; these effects were completely reversed by zinc chelator TPEN (20μM) added in the presence of ZnPy. Fluorescent zinc imaging with FluoZin3TM in HEK293 cells and DRG neurons demonstrated that ZnPy induced robust intracellular zinc rises which were completely reversed by TPEN. Four other chemically unrelated zinc ionophores PDTC, DEDTC, DIQ and Zinc ionophore I had similar to ZnPy effects: all four compounds augmented KCNQ4 and KCNQ2/3 currents and induced intracellular zinc rises; TPEN completely abolished these effects. Using voltage sensitive phosphatase CiVSP we found that the Ci-VSP-dependent PIP2 hydrolysis strongly inhibited recombinant KCNQ4 and KCNQ2/3, however, ZnPy and PDTC completely abolished these effect rendering channels insensitive to Ci-VSP activation. TIRF imaging using optical PIP2 reporter PLCδPH-GFP confirmed that neither ZnPy nor PDTC affected the ability of CiVSP to hydrolyze PIP2, as determined by the depolarization-induced PLCδPH-GFP translocation. In sum, our data suggest that intracellular zinc potently augment KCNQ channel activity by reducing channel requirement for PIP2.