Dataset Information

Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

ABSTRACT: The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent ?D and ?H strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the ?H and ?I strands of the adjacent subunit. Because the ?H strand forms a ? sheet with the ?I and ?D strands, the immobilization of the pore-forming ? sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.

SUBMITTER: Inanobe A

PROVIDER: S-EPMC3823594 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

Inanobe Atsushi A Nakagawa Atsushi A Kurachi Yoshihisa Y

PloS one 20131111 11

The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu2 ...[more]

PMID: 24244570

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Project description:Inward rectifier potassium (Kir) channels act as cellular diodes, allowing unrestricted flow of potassium (K(+)) into the cell while preventing currents of large magnitude in the outward direction. The rectification mechanism by which this occurs involves a coupling between K(+) and intracellular blockers-magnesium (Mg(2+)) or polyamines-that simultaneously occupy the permeation pathway. In addition to the transmembrane pore, Kirs possess a large cytoplasmic domain (CD) that provides a favorable electronegative environment for cations. Electrophysiological experiments have shown that the CD is a key regulator of both conductance and rectification. In this study, we calculate and compare averaged equilibrium probability densities of K(+) and Cl(-) in open-pore models of the CDs of a weak (Kir1.1-ROMK) and a strong (Kir2.1-IRK) rectifier through explicit-solvent molecular-dynamics simulations in ~1 M KCl. The CD of both channels concentrates K(+) ions greater than threefold inside the cytoplasmic pore while IRK shows an additional K(+) accumulation region near the cytoplasmic entrance. Simulations carried out with Mg(2+) or spermine (SPM(4+)) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K(+) in both channels. The results also show that SPM(4+) behaves differently inside these two channels. Although SPM(4+) remains inside the CD of ROMK, it diffuses around the entire volume of the pore. In contrast, this polyatomic cation finds long-lived conformational states inside the IRK pore, interacting with residues E224, D259, and E299. The strong rectifier CD is also capable of sequestering an additional SPM(4+) at the cytoplasmic entrance near a cluster of negative residues D249, D274, E275, and D276. Although understanding the actual mechanism of rectification blockade will require high-resolution structural information of the blocked state, these simulations provide insight into how sequence variation in the CD can affect the multi-ion distributions that underlie the mechanisms of conduction, rectification affinity, and kinetics.

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.

Dataset Information

Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

Publications

Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

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