Structural determinants of gating in inward-rectifier K+ channels

The gating characteristics of two ion channels in the inward-rectifier K+ c hannel superfamily were compared at the single-channel level. The strong in ward rectifier IRK1 (Kir 2.1) opened and closed with kinetics that were slo w relative to those of the weakly rectifying ROMK2 (Kir 1.1b). At membrane potential of -60 mV, both IRK and ROMK had single-exponential open-time dis tributions, with mean open times of 279 +/- 58 ms (n = 4) for IRK1 and 23 /- 1 ms (n = 7) for ROMK, At -60 mV (and no EDTA) ROMK2 had two closed time s: 1.3 +/- 0.1 and 36 +/- 3 ms (n = 7). Under the same conditions, IRK1 exh ibited four discrete closed states with mean closed times of 0.8 +/- 0.1 ms , 14 +/- 0.6 ms, 99 +/- 19 ms, and 2744 +/- 640 ms (n = 4), Both the open a nd the three-shortest closed-time constants of IRK1 decreased monotonically with membrane hyperpolarization; IRK1 open probability (P-o) decreased sha rply with hyperpolarization due to an increase in the frequency of long clo sed events that were attributable to divalent-cation-blockade. Chelation of divalent cations with EDTA eliminated the slowest closed-time distribution of IRK1 and blunted the hyperpolarization-dependent fall in open probabili ty. In contrast, ROMK2 had shorter open and closed times and only two close d states, and its P-o was less affected by hyperpolarization. Chimeric chan nels were constructed to address the question of which parts of the molecul es were responsible for the differences in kinetics. The property of multip le closed states was conferred by the second membrane-spanning domain (M2) of IRK. The long-lived open and closed states, including the higher sensiti vity to extracellular divalent cations, correlated with the extracellular l oop of IRK, including the "P-region." Channel kinetics were essentially una ffected by the N- and C-termini, The data of the present study are consiste nt with the idea that the locus of gating is near the outer mouth of the po re.