Figure 3.
Mechanism of the PIP2-induced loss of tolbutamide block. (A) Representative current recorded from inside-out membrane patches containing wild-type KATP channels exposed to differing [tolbutamide] or 30 μM ATP, as shown. The gaps in the record are 4.5, 2, and 1 min. The dashed line indicates zero current (determined in 5 mM ATP). (B) Plot of relative current (Irel) versus [tolbutamide] for trace segments shown in A, both before (•) and with time after (○) Pip2 application. Data at 10 mM tolbutamide are from control and 10-min time points. Fitted lines correspond to a least squares fit of the sum of two Hill equations as described in Fig. 1with the fraction of high affinity inhibition allowed to vary (37, 23, 13, 4, 0% at 0, 4.5, 7, 8 and 10 min) as indicated, after PIP2 application. The insert shows data only at 7 min. The dashed lines correspond to (A, dashed) the same curve as above, and (B, dotted) a least squares fit of the sum of two Hill equations, with the fraction of high affinity inhibition held constant at 37%, but with the affinity (i.e., K 1/2) allowed to vary. It is clear that high affinity block disappears because the high affinity fraction disappears, not because there is a change in the sensitivity of this component.