Rundown induced by 1 mM Mg²⁺ enhances the difference between WT and E128K response to PIP₂ stimulation. (A) Representative inside-out patch voltage clamp recordings from COSm6 cells transfected with WT (top) or E128K (bottom) K_ATP channels. Patches were pulled into Kint plus 1 mM MgCl₂ for 1 min to induce rundown to accentuate the differential response between WT and E128K channels; EDTA was not included in any of the solutions. Patches were then exposed to either 1 mM ATP or 5 µM PIP₂. Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 100 pA. Zero current is indicated by the dotted lines. Time of patch excision is noted by the arrow. (B) Change in current is expressed as the average current at each experimental time point relative to the initial maximum immediately upon patch excision. The change in current was determined after rundown and after one, two, or three 30-s exposures to 5 µM PIP₂; the point of each record analyzed is indicated by the numbers above each trace in A. Error bars represent SEM. n = 5–8 for each data point (*, P < 0.05; Student’s t test comparing WT and E128K for each point analyzed).