Fowler PW et al. (2014), Insights into the structural nature of the tran...

XB-ART-51324

Channels (Austin) 2014 Jan 01;86:551-5. doi: 10.4161/19336950.2014.962371.

Show Gene links Show Anatomy links

Insights into the structural nature of the transition state in the Kir channel gating pathway.

Fowler PW , Bollepalli MK , Rapedius M , Nematian-Ardestani E , Shang L , Sansom MS , Tucker SJ , Baukrowitz T .

???displayArticle.abstract???
In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics.

???displayArticle.pubmedLink??? 25483285
???displayArticle.pmcLink??? PMC4594414
???displayArticle.link??? Channels (Austin)
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: kcnj1 kcnj6

???attribute.lit??? ???displayArticles.show???

Figure 1. Mutations at several locations within the gating network differentially affect the pH-gating kinetics. (A) Location of the TM1, slide helix and G-loop mutations in the Open-Kir3.2 model. (B) Kinetics of pH-induced channel inhibition (pH 10 → pH 5) and pH recovery (pH 5 → pH 10) measured for WT and mutant channels. (C) Fold change in pH recovery (off-rate, red) and pH inhibition (on rate, gray) for TM1 and slide helix mutations (left), and G-loop mutations (right). The dotted line indicates the speed of the Na+ to K+ solution exchange for WT Kir1.1.

Figure 2. Gating kinetics provide insight into structure of the transition state. Free energy (ΔG) diagrams based on kinetic data for the closing (pH 10 → 5) and opening reactions (pH 5 → 10). These depict the energetic situation subsequent to the protonation and deprotonation steps. PO*, TS* and C* represent the relative free energies after point mutations are introduced in either TM1 and slide helix (A), or in the G-loop (B). Upper gray boxed panels indicate the change in free energy for the indicated states caused by the mutations; note that mutations in TM1 and slide helix (A) increase the free energy of the PO* and TS* states equally, but to a lesser extent the C* state (because the gating network is missing). This results in increased pH-sensitivity due to unchanged inhibition rates and slower activation rates. By contrast, G-loop mutations have relatively little effect on the TS* state (due to an unstructured G-loop). Instead, both the PO* and C* states are affected (B). However, the destabilising effect on the PO* state is greater than on the C* state leading to a faster rate of inhibition and thus an overall increased pH-sensitivity. (C) Cartoon depicting the structural and thermodynamic changes in Kir1.1 upon pH-inhibition. At pH 10, Kir1.1 exists predominantly in the open state (green residues indicate the gating network in this state). Protonation (pH 5) causes an increase in the free energy of the open state and pre-open states (blue residues indicate the gating network in this state) favoring the transition into the more stable closed state (red residues indicate the defragmented gating network). Kinetic analysis of mutants shown in Figure 1 suggests that the structure of the rate determining transition state most closely resembles the pre-open state (gating network shown in blue), but with a different G-loop structure (yellow). This altered G-loop structure appears to be less sensitive to mutagenic destabilization than the open, pre-open or closed state.

References [+] :

An, The cytosolic GH loop regulates the phosphatidylinositol 4,5-bisphosphate-induced gating kinetics of Kir2 channels. 2012, Pubmed

An, The cytosolic GH loop regulates the phosphatidylinositol 4,5-bisphosphate-induced gating kinetics of Kir2 channels. 2012, Pubmed
Bollepalli, State-dependent network connectivity determines gating in a K+ channel. 2014, Pubmed , Xenbase
Choe, A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating. 1997, Pubmed , Xenbase
Hansen, Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. 2011, Pubmed
Meng, The molecular mechanism by which PIP(2) opens the intracellular G-loop gate of a Kir3.1 channel. 2012, Pubmed
Paynter, Random mutagenesis screening indicates the absence of a separate H(+)-sensor in the pH-sensitive Kir channels. 2010, Pubmed
Pegan, Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification. 2005, Pubmed , Xenbase
Rapedius, H bonding at the helix-bundle crossing controls gating in Kir potassium channels. 2007, Pubmed , Xenbase
Rapedius, Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing. 2007, Pubmed
Sadovsky, Principles underlying energetic coupling along an allosteric communication trajectory of a voltage-activated K+ channel. 2007, Pubmed
Tao, Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution. 2009, Pubmed , Xenbase
Whorton, Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium. 2011, Pubmed , Xenbase
Yifrach, Energetics of pore opening in a voltage-gated K(+) channel. 2002, Pubmed , Xenbase