Figure 1.

Identification of E229A as an inactivation mutant. (A) WT channel activities recorded in inside-out membrane patches using K-INT (left) or K-INT + 1 mM EDTA (right) bath solution. Arrows indicate patch excision. Channel rundown is dramatically reduced by the addition of 1 mM EDTA to the bath solution. (B) Using K-INT/EDTA solution, inactivation is observed in the E229A mutant channels. Representative currents recorded from inside-out membrane patches containing WT, the E229A, or the R314A mutant channels are superimposed for comparison. The arrow indicates patch excision into K-INT/EDTA. The kinetics of inactivation of the E229A channels are similar to the R314A channels. The inactivation time course of both E229A and R314A channels was fitted by a single exponential (indicated by the black curves). (C) Channels formed by WT-Kir6.2ΔC25 in the absence of SUR1 exhibited stable activities after patch excision into K-INT/EDTA (left). In contrast, channels formed by E229A-Kir6.2ΔC25 (middle) or R314A-Kir6.2ΔC25 (right) in the absence of SUR1 showed rapid current decay. Currents in this and subsequent figures were recorded at −50 mV membrane potential. Inward currents are shown as upward deflections.