Many ion channels and transporters are regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂) in the cell membrane. There is evidence that cardiac slow delayed rectifier K⁺ current I_Ks, composed of KCNQ1 and the β-subunit KCNE1, is regulated by PIP₂ via KCNE1. It is controversial, however, whether KCNQ1/KCNE1 channel currents are negatively or positively regulated by PIP₂^1,2_. Important molecular tools to study ion channel regulation by PIP₂ in living cells are FRET-sensors for monitoring changes in the abundance of PIP₂ and voltage-sensing PI-phosphatases (VSPs) for controlled depletion independent of cellular signaling pathways. We used a tricistronic (2A-peptide) vector³ for simultaneous expression of two PH-domains of PLC_δ1 fused to CFP and YFP respectively, serving as FRET sensors and Ci-VSP, in a stable KCNQ1/KCNE1 CHO cell line. Transfection with this vector caused a basal reduction of whole-cell I_Ks density, independent of activation of Ci-VSP. Activation of Ci-VSP by voltage steps to + 60 mV resulted in transient depletion of PIP₂, resulting in slowed activation, accelerated deactivation, and reduction of I_Ks current density. Upon repetitive activation of Ci-VSP, two components of inhibition were observed. One component was directly related to the transient reduction in membrane PIP₂ indicated by the FRET signal. The second component, unrelated to the FRET signal, was slowly reversible resulting in a progressive reduction in current during repetitive (0.06 s^-1) activation of Ci-VSP. Both components were abolished by supplementing the pipette filling solution with diC8-PIP₂. No evidence for a negative regulation of I_KS by PIP₂ was obtained in these experiments.

¹Li et al. (2011) PNAS, 31;108:9095-10000;

²Ding et al. (2004) JBC, 279: 50726-50734;

³Hertel et al. (2011), PlosOne, 6: e20855