Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification

Abstract

N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two new crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1(L)) and the G protein-sensitive Kir3.1 (Kir3.1(S)) channels in the absence of PIP(2) show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1(L) and Kir3.1(S) suggests a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structural comparison shows a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1(L) that is important for modulating inward rectification. Taken together, these results suggest the cytoplasmic domains of Kir channels undergo structural changes to modulate gating and inward rectification.

Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel

“…2 A and B). ␤-Sheets at equivalent locations have been identified in the crystal structures of inward rectifier K ϩ channels (Kir channels) (11)(12)(13)(14) and a cyclic nucleotidemodulated (HCN2) channel (15) (Fig. 2C and Fig.…”

NMR Studies of Interactions between C-Terminal Tail of Kir2.1 Channel and PDZ1,2 Domains of PSD95

“…Indeed, the observed cross-peaks in the TROSY spectrum of 15 side chain identity, we assigned 44 distinct peaks of the 51 possible residues in the tail, resulting in 86% coverage of the possible N-H moieties. (Figure 2a) (20,22 13 C R chemical shift analysis shows on average low divergence from the values of a random coil indicating a lack of secondary structure for this region (Figure 2b). Only, the C-terminal residue (I428) had a positive shift, which is attributed to the influence of the additional carboxyl group of the C-terminus.…”

“…GST was fused directly to the C-terminal tail of Kir2. 1 (354-428) 13 C-labeled samples were obtained by expressing in bacteria grown at 37°C to 0.6 OD in LB media. Cells were spun down, washed with PBS, spun again, and resuspended in minimal media containing appropriate isotope labels (15).…”

“…A structure of this region of Kir2 channels with its cognate PDZ domain could help reveal a more detailed mechanism of binding specificity. Recent structural studies utilizing a N-C terminal fusion that contained the entire C-terminal end of Kir2.1, Kir2.1 L (44-64 fused to 189-428), was only visualized up to residue 371, suggesting that the remaining 57 C-terminal tail amino acids were disordered ( Figure 1c) (13). In order to determine the exact region of the Kir2 C-terminal segment involved in binding PDZ domains, we have characterized the interactions of Kir2.1 with the PDZ1 and PDZ2 domains of PSD95 by nuclear magnetic resonance (NMR) spectroscopy.…”

Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes

“…2c). E292 also lies within one of the two gating loops that link the ATP-binding site to the slide helix, and which may, therefore, be involved in coupling ATP binding to channel opening [35,40]. Thus, disruption of the putative E292-R301 ion pair can be hypothesized to either alter the mechanism by which ATP binding is linked to channel gating, or to destabilize ATP binding indirectly, by altering the conformation of the ATP-binding site.…”