Figure 1.

PIP₂ mimics the MgATP dependence of the rundown of G protein-mediated activation as well as channel sensitization to gating by internal Na⁺ ions. (A) NP_o plot of K_ACh channel activity in an inside-out patch from a chicken embryonic atrial myocyte, showing that in the presence of ATP (2 mM, sodium salt, in the presence of 0.6 mM Mg²⁺ in the bath solution), GTP (100 μM) produced sustained activity (10 μM acetylcholine was present in the pipette). Withdrawal of ATP caused a gradual rundown of the GTP-induced activity. As can be seen at the beginning of the record ATP perfusion alone did not activate the channel. Application of each nucleotide is indicated by the bars. Representative single-channel currents are shown before and after the rundown of the activity, as indicated at time points a and b during the course of the experiment. (B) Plot similar to that in A, showing that PIP₂ restored the ability of GTP to stimulate channel activity and mimicked ATP in its ability to prevent rundown of the GTP-induced channel activity. Acetylcholine at 5 μM was present in the pipette solution. Perfusion of the inside-out patch with GTP (100 μM) and PIP₂ (5 μM) in the bath solution is indicated by bars. The arrows labeled a and b show time points during which rundown and restored single channel activity is shown, respectively. PIP₂, like ATP, did not stimulate channel activity when perfused alone. (C) NP_o and MT_o plots of K_ACh channel activity in inside-out patches from Xenopus oocyte coinjected with GIRK1/GIRK4 cRNAs. In the absence of MgATP, the channels did not respond to Na⁺ stimulation (20 mM). MgATP (5 mM) modified channels to a longer-lived open state but produced little activation. After this effect, channels responded to Na⁺ by increasing NP_o with no further significant change in MT_o. (D) Experiment similar to that in A from another patch showing that PIP₂ (1 μM) produced similar effects on MT_o and sensitization to gating by intracellular Na⁺ (20 mM). PIP₂ application, unlike ATP, had long lasting effects and, therefore, it was not necessary to apply it with Na⁺ ions for maximal effects.