Figure 3.

Depletion of PIP₂ by PLCs blocks MgATP and G_βγ stimulation of K_ACh channel activity and restoration of PIP₂ reverses these effects. (A) Single-channel NP_o and MT_o plots as a function of time in the experiment were obtained from an inside-out patch of a Xenopus oocyte expressing GIRK1/GIRK4. The K_ACh channels were first activated by application of MgATP/Na (5/20 mM). Both NP_o and MT_o were increased by MgATP/Na. Subsequent treatment of the inside-out patch with phosphatidylinositol-specific PLC (1 unit/ml) for approximately 2.5 min, significantly reduced activation by MgATP/Na, causing a concomitant decrease in MT_o. (B) Single-channel NP_o and MT_o plots, obtained from an inside-out patch of a Xenopus oocyte expressing GIRK1/GIRK4. Soon after excision, G_βγ application (20 nM) caused persistent channel activation. Subsequent treatment of the patch with PLC-β2 (5 μg/ml) inhibited activity with a concomitant decrease in MT_o. (C) NP_o plot as a function of time in the experiment from an inside-out patch of Xenopus oocyte expressing GIRK1/GIRK4. The patch was exposed to PLC-β2 (5 μg/ml) for a period greater than 5 min, before G_βγ (20 nM) application. PLC-β2 treatment greatly retarded G_βγ effectiveness. Subsequent perfusion with PIP₂ (5 μM) revealed high channel activity, thus rescuing the G_βγ action. The number of active K_Ach channels in the membrane was greater than 5, thus precluding analysis of MT_o [see Sui et al. (13)]. (D) Single-channel NP_o and MT_o plots, obtained from an inside-out patch of Xenopus oocyte expressing GIRK1/GIRK4. Perfusion of the patch with ATP-free solutions for several minutes rendered G_βγ (20 nM) ineffective. Subsequent addition of MgATP (5 mM) to the membrane revealed high channel activity, thus rescuing the G_βγ action, which was presumably still bound but ineffective.