The majority of ion channel species, including Kir3.x, which carries the cardiac muscarinic K⁺ current (I_K(ACh)), is sensitive to changes of PIP₂ in the plasma membrane. As precursor of the second messengers IP₃ and DAG, PIP₂ is depleted by activation of PLC, resulting in superposition of effects related to PIP₂ and signals downstream of IP₃ and DAG. In order to distinguish different mechanisms of regulation of I_K(ACh) by endogenous G_q-coupled a₁ receptors in atrial myocytes, we used a multicistronic 2A peptide based adenoviral vector for simultaneous manipulation and monitoring of PIP₂ plasma membrane levels. The vector encodes for three proteins form a single ORF: Ci-VSP, a voltage-activated phosphatase, which dephosphorylates PI-(4,5)-bisphosphate (PIP₂) and a PIP2-sensitive FRET pair consisting of PH-domains of PLC_d1 fused to either CFP or YFP. In isolated adult rat atrial myocytes infected with this construct, endogenous I_K(ACh) and CFP/YFP FRET ratio recorded simultaneously were reduced by depolarizing voltage steps in a graded voltage- and time-dependent fashion. Activation of the G_q signaling pathway by a₁-AR resulted in an inhibition of I_K(ACh), which was only partially related to PIP₂ depletion indicated by the FRET measurement. Direct activation of PKC by phorbolester resulted in inhibition of I_K(ACh) without a change in membrane PIP₂. From these data it can be estimated that the inhibition of I_K(ACh) upon a₁-AR activation is due to PIP₂ depletion by about 50%. Moreover, activation of PKC resulted in an increase in the halftime of recovery of current subsequent to Ci-VSP-induced PIP₂ depletion, whereas recovery of the FRET signal was not affected. This behaviour can be modelled by assuming that phosphorylation by PKC results in a reduced PIP₂-affinity of Kir3 channel complexes.