Figure 3.

Enhancement of PIP₂ activation of the channels by PKA phosphorylation. In each experiment, channels were excised and allowed to run down in Mg²⁺ solution. The horizontal time bar is 120 sec. (A) Mg-ATP[γS] (0.5 mM in Mg²⁺ solution) did not activate channels after run-down in Mg²⁺ solution. Subsequent addition of Mg-ATP (0.5 mM) activated the channels. (n = 3.) (B) Although PKA catalytic subunit (100 units/ml) + Mg-ATP[γS] (0.5 mM) did not activate the channels, it apparently increased the rate of channel activation by ATP and reduced the sensitivity of the ATP-activated channels to inhibition by anti-PIP₂ antibodies (40 nM). (n = 3.) (C) Dose–response curves (assuming one binding site) for PIP₂ (0.03–50 μM) activation of the channels with or without pretreatment by PKA + ATP[γS]. Currents activated by PIP₂ (at −30 mV holding potential) are shown as percentage of on-cell currents. As the PIP₂ concentration in the membrane is likely different from that calculated based on the amount of PIP₂ dispersed in aqueous solution (as shown here), it is difficult to compare the half-maximal concentration for PIP₂ activation of the channels to the K_d determined from the in vitro binding of ³H-PIP₂ to GST–RKC (see Fig. 1 C and D).