Figure 1.

Brain PIP₂ and soluble PIP₂ activate reconstituted brain GIRK2 channels in the absence of other proteins. (a) Graph shows purification of mouse GIRK2 from Pichia pastoris. The absorbance is plotted as a function of elution volume, with molecular weight (MW) standards; Blue - 440 kDa, Black - 158 kDa, Red - 75 kDa (arrows). The major peak elutes at a volume consistent with the size of a GIRK2 tetramer (T). Inset, Coomassie blue staining of a gradient SDS-PAGE protein gel shows two bands, one for tetramer (T) and one for monomer (M). Inset, wild-type GIRK2 was truncated at the amino- and carboxy-termini (see methods). (b) Example of K⁺ flux assay with GIRK2 reconstituted into PE/PG liposomes in the absence or presence of brain PIP₂ (1%) (see methods). The fluorescence is normalized and plotted as a function of time (‘Relative K⁺ flux’). Arrows indicate addition of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the GIRK2 channel blocker MTS-HE (HE, 100 μM) and valinomycin (VM). Note the quenching of fluorescence for GIRK2 with PIP₂ (green) upon addition of CCCP, which is attenuated with addition of MTS-HE. Dashed line represents no flux. (c) Representative fluorescent traces of GIRK2 alone (grey), GIRK2 and MTS-HE (red), and GIRK2 with acute application of 30 μM diC₈-PIP₂ (green). The decay was fit with a single exponential to determine that rate constant (1/τ, s^-1). (d) Bar graph shows the average ( ± SEM) fractional activation with diC₈-PIP₂ based on the change in steady-state fluorescence (green bar vs. grey bar). MTS-HE (red bar) significantly inhibits diC₈-PIP₂-activated GIRK2 channels (n = 7, P < 0.0001).