Fig. 4. Biophysical and functional assays on Kir2.1 WT and R312H mutant.

(A and B) Comparative binding kinetics of PIP₂ on Kir2.1 WT (A) and R312H mutant (B) immobilized on a CM5 chip. Five sequential injections of PIP₂ are shown, 1.25 to 20 μM, at 25 μl/5 min. In color, experimental data; in black, fitted curve. The single-cycle kinetics assays were carried out in triplicate; only one replica is shown. The SPR response is expressed in response units relative to the time in seconds. (C and D) Electrophysiological recordings of Kir2.1 WT (C) and R312H (D) reconstituted in lipid bilayers in the presence of 1% PIP₂ at −100 mV. Kir2.1 WT shows an open probability (Po) of 70%, and Kir2.1 R312H has a Po of less than 1%.