G protein-gated inwardly rectifying K+ channels (Kir3.x. GIRK) are characteristically activated through binding βγ subunits of G_i/o proteins. It is thought that the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), is critical for Kir3.x channel activation (Huang et al., 1998). Stimulation of G_q/11-coupled receptors results in downstream activation of phospholipase C and consequently PIP₂ metabolism. Using the M₃ receptor, we sought to investigate the influence that potential PIP₂ depletion has on the activities of the neuronal, Kir3.1/3.2A, and atrial, Kir3.1/3.4, isoforms. Perforated patch (Kir3.1/3.2A), whole-cell (Kir3.1/3.4) and single channel (inside-out) patch clamp was used to record membrane currents under symmetrical K⁺ conditions (~140 mM K⁺,ATP/GTP supplementation as appropriate) in HEK293 cells. Kir3.1/3.2A channels were stably expressed along with either muscarinic M₃ or adenosine A₁ receptors. Kir3.1/3.4 channels were stably expressed and the desired receptor was expressed transiently. Data are presented as mean±SEM. One-way ANOVA was used to test for statistical significance.We have previously demonstrated that Kir3.1Kir3.2A currents are essentially irreversibly inhibited in the whole-cell configuration after M₃ receptor activation. We next examined if this was dependent on the channel or configuration of the patch clamp used. In whole cell recordings from Kir3.13.4 expressing cells, M₃ receptor stimulation (10µM carbachol, CCh) resulted in an initial potentiation (basal: 61±4 pA/pF, +CCh: 93±5 pA/pF) followed by a significant inhibition (36±3 pA/pF, n=10, p<0.001). Recovery was complete within 8 minutes (54±6pA/pF). Using the perforated patch configuration, Kir3.1/3.2A channel activity underwent a similar pattern of modulation (basal: 23±4.4 pA/pF, +CCh: 39±6 pA/pF, inhibition: 8±2 pA/pF p<0.05, recovery at 5 min: 21±5, n=12). Recovery from inhibition for both heteromeric complexes was prevented by 10 minutes preincubation with 10µM wortmannin prior to receptor activation. Single-channel current amplitudes showed a strong inward-rectification with a unitary conductance of 32±2pS (n=8) for Kir3.1/3.2A and 33±1pS (n=3) for Kir3.1/3.4. Single-channel open probability (NPo) increased and was well maintained after application of 1µM PIP₂ applied to the intracellular face of the patch (Kir3.1/3.2A/A₁, no receptor stimulation: NPo 0.038±0.02 ATP/GTP, 0.685±0.08 PIP₂/GTP n = 4; Kir3.1/3.4: NPo 0.013±0.002 ATP/GTP, 0.074±0.011 PIP₂/GTP n=3). Our data demonstrate the dependence of GIRK channel activity on PIP₂ and show that replenishment of anionic phospholipids is necessary for recovery from inhibition after phospholipaseCβ activation.