A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel
Abstract: G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+, while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+, suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+-selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q sensitive K+ current and the second a TPN-Q resistant Li+ current. The results show that a novel Li+ permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix (PH), S148F and T151A, also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations. Significance Statement Kir3.2 G156S is a Na+-permeable inherited mutant which causes neurological disorders in mammals. The structural changes underlying the loss of K+ selectivity induced by this inherited mutation remain unknown. Here we show evidences revealing a novel mechanism underlying the abnormal ion selectivity that Kir3.2 G156S has two ion permeation pathways, the conventional SF route which permeates K+ predominantly and a novel route which permeates Li+ and Na+ preferentially. This provides us with information for the design of new effective drugs for disease treatment at the early stages, which can selectively block only the novel cation permeation pathway.
Summary (3 min read)
- These amino acid residues are conserved among GIRK subtypes.
- The structural changes induced by mutations at different regions of the channel which alter the ion selectivity still remain to be elucidated.
- No further experimental data have been provided to support these hypotheses.
Different disease associated mutations of GIRK channels show diverse ion selectivity
- To characterize the ion selectivity changes of GIRK channels induced by inherited mutations, the authors constructed these mutants using mouse Kir3.2 or rat Kir3.4 channels .
- In the case of GIRK channels which show strong rectification, however, the reversal potential is significantly affected by even a small leak current which hinders accurate analysis of PX / PK.
- The information the authors present in this study to compare the ion selectivity is the profile of sequence of current amplitudes which reflects the extent of the permeation of various ions.
- Since the homotetramers of the Kir3.4 SF mutant G151R showed very small currents from all extracellular cations, ion permeation could not be compared because of contamination from endogenous currents .
- A previous study suggested that this may be relevant to the eliminated hydrogen bonds of Thr158 with Pro128, Cys129 and Lys160, which are conserved among GIRK subtypes (Choi et al., 2011) .
Effect of TPN-Q on the ion selectivity of Kir3.2 G156S
- To further clarify whether a novel ion permeation pathway besides the conventional SF route does exist in Kir3.2 G156S, the authors examined the effect of another pore blocker which binds to the GIRK channel at a different position than Ba 2+ .
- Here the authors used Tertiapin-Q (TPN-Q) known to specifically block Kir1.1 and GIRK channels by capping the SF pore from the extracellular side (Doupnik, Parra, & Guida, 2015; Patel, Kuyucak, & Doupnik, 2020) .
- Taken together, when the SF pathway was blocked by TPN-Q, a high PLi/PK was observed presumably due to the novel ion permeation route.
Activation effect of Gβγ and Ivermectin on Kir3.2 G156S
- Previous studies showed that heteromeric channels comprised of Kir3.2 G156S and Kir3.1 are insensitive to Gβγ-stimulation (Navarro et al., 1996) .
- Here the authors examined whether homomeric channels of Kir3.2 G156S are also insensitive to the Gβγstimulation by coexpressing Kir3.2 G156S with the muscarinic M2 receptor (M2R).
- Applications of 10 μM IVM induced the current increase in both the Kir3.2 WT and G156S mutant.
- This shows that the limited response of the G156S mutant to ACh stimulation is not due to the high basal activity reaching a saturation level, since IVM can further increase the current amplitude.
- Taken together, the G156S mutation may induce a conformational change that occurs not only in the SF region but also in the structural components which play roles in the Gβγ-coupling-channel activation linkage.
Single-channel recordings of Kir3.2 G156S show two types of ion conducting events
- When 400 nM TPN-Q was added to the pipette solution, these events with a relatively large current amplitude were largely absent and replaced by infrequently observed events with a very small current amplitude (< 0.2 pA) .
- Occasional amplifier resets were unavoidable during these long recordings due to the need to reset the headstage output periodically as part of the capacitive feedback system used to achieve low noise recordings .
- 5a. Inclusion of 400 nM TPN-Q in the pipette did not apparently change the activity and the amplitude of Li + current , showing that the major events caused by Li + influx through Kir3.2 G156S were not blocked by TPN-Q .
- These results demonstrate that two types of ion conducting events are present in the same recording of Kir3.2 G156S.
- This supports that a novel ion permeation pathway in addition to the SF route is formed by the G156S mutation in the Kir3.2 channel.
- This novel ion permeation route is similar to a side entry pathway for Na + permeation through the sodium potassium (NaK) channel (Roy et al., 2021; Shi et al., 2018) .
- The sequence of ion preference of the G156R and L173R mutants (Rb + , Cs + -preferable permeation) fits to I -IV of the Eisenman sequence, suggesting that the modified SF pathway is a weak-field-strength site type, where the site-interaction energy is less than the hydration energy (Eisenman, 1962; Hille, 2001) .
- Further detailed analysis was not performed due to the difficulty of the precise estimation of the small current amplitudes of fast gating events.
- In conclusion, their results reveal the presence of a novel ion permeation pathway besides the SF route in Kir3.2 G156S.
- The design of drugs that selectively block the novel cation permeation route alone to eliminate the abnormal cation fluxes may have the potential to develop a novel treatment for Kir disorders such as for KPLBS patients with the Kir3.2 G154S mutation (corresponding to mouse G156S) at an early stage of life.
Mutagenesis and cRNA preparations
- The authors constructed mutants of Kir3.2 and Kir3.4 and prepared their cRNA as described previously (Chen et al., 2019) .
- By PCR using PfuUltra II Fusion HS DNA Polymerase kit (Agilent technologies, Santa Clara, CA, USA) and the primers (Supplementary Table 1 ), the authors introduced point mutations in mouse Kir3.2 (GenBank accession no.: AF040051) or rat Kir3.4 (GenBank no.: L35771) which were confirmed by DNA sequencing.
- Their cDNA were linearized by restriction enzymes, and the complementary RNA were transcribed by the mMessage T3 or T7 mMachine Kit (Ambion, Austin, TX, USA).
- The authors purchased adult female Xenopus laevis from Hamamatsu Seibutsu Kyouzai (Hamamatsu, Japan).
- Isolation of oocytes from the frogs were performed as described previously (Chen et al., 2019) .
- After removing the follicles, each oocyte was injected with 50 nl of cRNA solution and then incubated in frog Ringer's solution with 0.1% penicillin-streptomycin (Sigma-Aldrich) at 17°C.
- The oocytes were used for twoelectrode voltage-clamp recordings 1-5 days after the injection of cRNA.
Two-electrode voltage-clamp recordings
- Glass electrodes for two-electrode voltage-clamp recordings had a resistance of 0.2-0.5 MΩ when filled with a pipette solution containing 3 M potassium acetate and 10 mM KCl.
- To evaluate the ion selectivity, the KCl was replaced by LiCl, NaCl, RbCl, CsCl, methylammonium-Cl, tetramethylammonium-Cl, tetraethylammonium-Cl, or NMDG-Cl, one at a time.
- Data were recorded by an oocyte clamp amplifier OC-725C (Warner instruments, Holliston, MA, USA), a digital converter Digidata 1440 (Molecular devices, San Jose, CA, USA), and pCLAMP 10 software (Molecular devices).
- Compounds were applied to the extracellular solution and perfused the oocytes in a recording chamber.
- Cell-attached recordings were carried out using ltk-mouse fibroblast cells as previously described (Eldstrom, Wang, Werry, Wong, & Fedida, 2015; Murray et al., 2016; Westhoff, Eldstrom, Murray, Thompson, & Fedida, 2019) .
- Briefly, cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as per the manufactures protocol, with DNA ratios of 1:0.8 of Kir3.2 G156S:GFP in micrograms.
- Tertiapin-Q (TPN-Q) was purchased from Abcam (Cambridge, UK) or Bio-Techne Canada (Toronto, Canada).
- ACh, Ivermectin (IVM), and the other reagents were purchased from Sigma-Aldrich (St. Louis, USA), unless otherwise specified.
- TPN-Q and ACh were dissolved in distilled water, and IVM was dissolved in DMSO.
- These reagents were diluted to a final concentration in the extracellular solution and the solvent concentration was ≤ 0.3%.
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