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Structural determinants of gating in inward-rectifier K+ channels.

ABSTRACT: The gating characteristics of two ion channels in the inward-rectifier K+ channel superfamily were compared at the single-channel level. The strong inward rectifier IRK1 (Kir 2.1) opened and closed with kinetics that were slow relative to those of the weakly rectifying ROMK2 (Kir 1.1b). At a membrane potential of -60 mV, both IRK and ROMK had single-exponential open-time distributions, 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 times: 1.3 +/- 0.1 and 36 +/- 3 ms (n = 7). Under the same conditions, IRK1 exhibited 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 and the three shortest closed-time constants of IRK1 decreased monotonically with membrane hyperpolarization. IRK1 open probability (Po) decreased sharply with hyperpolarization due to an increase in the frequency of long closed 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 probability. In contrast, ROMK2 had shorter open and closed times and only two closed states, and its Po was less affected by hyperpolarization. Chimeric channels were constructed to address the question of which parts of the molecules were responsible for the differences in kinetics. The property of multiple closed states was conferred by the second membrane-spanning domain (M2) of IRK. The long-lived open and closed states, including the higher sensitivity to extracellular divalent cations, correlated with the extracellular loop of IRK, including the "P-region." Channel kinetics were essentially unaffected by the N- and C-termini. The data of the present study are consistent with the idea that the locus of gating is near the outer mouth of the pore.

SUBMITTER: Choe H

PROVIDER: S-EPMC1300174 | biostudies-other | 1999 Apr

REPOSITORIES: biostudies-other

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Structural determinants of gating in inward-rectifier K+ channels.

Choe H H Palmer L G LG Sackin H H

Biophysical journal 19990401 4

The gating characteristics of two ion channels in the inward-rectifier K+ channel superfamily were compared at the single-channel level. The strong inward rectifier IRK1 (Kir 2.1) opened and closed with kinetics that were slow relative to those of the weakly rectifying ROMK2 (Kir 1.1b). At a membrane potential of -60 mV, both IRK and ROMK had single-exponential open-time distributions, 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 ...[more]

PMID: 10096896

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Project description:Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.

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Structural determinants of gating in inward-rectifier K+ channels.

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Structural determinants of gating in inward-rectifier K+ channels.

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