Figure 6. Concentration dependence of GIRK2 activation by C8- and brain-PIP2.
(A) Current recorded from a bilayer containing GIRK2 and Gβγ during local perfusion of C8-PIP2 (black bars above recording) or buffer (white bars above recording). (B) Plot of current recorded from bilayers with GIRK2 and Gβγ (normalized to current at 28 μM C8-PIP2, mean ± SEM, n = 3 bilayers) vs concentration of C8-PIP2. Hill fit (solid line) gives an apparent dissociation constant of ∼15 μM and a Hill coefficient of ∼3.1. Fit to a non-cooperative model (‘Materials and methods’) in which simultaneous binding of 4 PIP2 molecules to one channel is required for channel opening is also shown (dotted line). (C) Current recorded from a bilayer with GIRK2, Gβγ and 1.38% (mole fraction) brain PIP2 during local perfusion of 32 μM C8-PIP2 (black bars above recording) or buffer (white bars above recording). Channels oriented with their extracellular side facing the PIP2 perfusion chamber were blocked with 100 nM TPNQ in perfusion buffers. As a way to normalize channel numbers in different membranes, the current value during perfusion of buffer was normalized to the current level during perfusion of 32 μM C8-PIP2. (D) Plot of current recorded from bilayers with GIRK2 and Gβγ (mean ± SEM, n = 3 bilayers) vs concentration of brain PIP2 in the membrane. Regression to Hill equation (solid line) resulted in an apparent dissociation constant of ∼0.8% mole fraction brain PIP2 and a Hill coefficient of ∼2.4. The dashed line shows regression to the same non-cooperative four sites model as in (B).