A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel

TL;DR: In this article, the authors investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants and showed that the mechanism underlying the loss of K+ selectivity induced by this inherited mutation involves formation of a novel ion permeation pathway besides the selectivity filter pathway.

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

Introduction

• 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.

Discussion

• 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).

Oocyte preparations

• 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.

Single-channel recordings

• 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.

Reagents

• 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%.

Full text

1
A novel ion conducting route besides the central pore in an 1
inherited mutant of G-protein-gated inwardly rectifying K
+
channel 2
3
I-Shan Chen
abc
*, Jodene Eldstrom
d
, David Fedida
d
, and Yoshihiro Kubo
ab
* 4
5
a
Division of Biophysics and Neurobiology, Department of Molecular and Cellular 6
Physiology, National Institute for Physiological Sciences, National Institutes of 7
Natural Sciences, Okazaki 444-8585, Japan 8
b
Department of Physiological Sciences, School of Life Science, SOKENDAI (The 9
Graduate University for Advanced Studies), Hayama 240-0193, Japan 10
c
Department of Pharmacology, School of Medicine, Wakayama Medical University, 11
Wakayama 641-8509, Japan 12
d
Department of Anesthesiology, Pharmacology and Therapeutics, University of 13
British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada 14
15
*Corresponding authors: I-Shan Chen, Yoshihiro Kubo 16
Email: chenis@wakayama-med.ac.jp; ykubo@nips.ac.jp 17
18
ORCIDs: I-S. C.: 0000-0003-3204-3921; J. E.: 0000-0002-7684-175X; 19
D. F.: 0000-0001-6797-5185; Y. K.: 0000-0001-6707-0837 20
21
Preprint: bioRxiv, DOI: https://doi.org/10.1101/2021.08.18.456735 22
23
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 11, 2021. ; https://doi.org/10.1101/2021.08.18.456735doi: bioRxiv preprint

2
Abstract
24
G-protein-gated inwardly rectifying K
+
(GIRK; Kir3.x) channels play important 25
physiological roles in various organs. Some of the disease-associated mutations of 26
GIRK channels are known to induce loss of K
+
selectivity but their structural changes 27
remain unclear. In this study, we investigated the mechanisms underlying the 28
abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-29
clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we 30
observed that Kir3.2 G156S permeates Li
+
better than Rb
+
, while T154del or L173R 31
of Kir3.2 and T158A of Kir3.4 permeate Rb
+
better than Li
+
, suggesting a unique 32
conformational change in the G156S mutant. Applications of blockers of the 33
selectivity filter (SF) pathway, Ba
2+
or Tertiapin-Q (TPN-Q), remarkably increased the 34
Li
+
-selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-35
channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of 36
events were observed, one attributable to a TPN-Q sensitive K
+
current and the 37
second a TPN-Q resistant Li
+
current. The results show that a novel Li
+
permeable 38
and blocker-resistant pathway exists in G156S in addition to the SF pathway. 39
Mutations in the pore helix (PH), S148F and T151A, also induced high Li
+
40
permeation. Our results demonstrate that the mechanism underlying the loss of K
+
41
selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway 42
besides the SF pathway, which allows permeation of various species of cations. 43
44
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 11, 2021. ; https://doi.org/10.1101/2021.08.18.456735doi: bioRxiv preprint

3
Introduction 45
G-protein-gated inwardly rectifying K
+
(GIRK) channels are stimulated by the 46
activation of G-protein-coupled receptors (GPCRs) to regulate heartbeat, neuronal 47
excitability and hormone secretion (Hibino et al., 2010). There are four types of GIRK 48
subunits (Kir3.1, Kir3.2, Kir3.3, and Kir3.4) which form homotetramers of Kir3.2 or 49
heterotetramers of multiple combinations in different tissues (Hibino et al., 2010; 50
Krapivinsky et al., 1995; Kubo, Reuveny, Slesinger, Jan, & Jan, 1993; Lesage et al., 51
1994), and gene mutations encoded by GIRK channels have been found to cause 52
diseases in some patients (Zangerl-Plessl, Qile, Bloothooft, Stary-Weinzinger, & van 53
der Heyden, 2019). For example, deletion of Thr152, or mutations of Gly154 to Ser or 54
Leu171 to Arg in the human Kir3.2 channel (encoded by KCNJ6), cause Keppen-55
Lubinsky syndrome (KPLBS) or KPLBS-like disorder (Horvath et al., 2018; Masotti et 56
al., 2015), and G151R, T158A or L168R mutations in the human Kir3.4 channel 57
(encoded by KCNJ5) cause aldosterone-producing adenomas (APA) (Boulkroun et 58
al., 2012; Choi et al., 2011). These amino acid residues are conserved among GIRK 59
subtypes. Some of the inherited mutations of these residues have been reported to 60
cause loss of K
+
selectivity, resulting in disorder of cell function and cell death (Choi 61
et al., 2011; Horvath et al., 2018; Navarro et al., 1996; Scholl et al., 2012; Slesinger 62
et al., 1996). 63
K
+
selectivity is achieved by a highly conserved amino acid sequence TV(I)GYG 64
which forms the selectivity filter (SF) in K
+
channels (Zagotta, 2006). Mutations in the 65
SF sequence of K
+
channels alter ion selectivity (Heginbotham, Lu, Abramson, & 66
MacKinnon, 1994). An ion pair, conserved in the Kir channel family, a glutamic acid 67
located in the pore helix (PH) and an arginine located in the extracellular loop behind 68
the SF, is known to contribute to stabilization of the pore structure and maintaining 69
ion selectivity (Yang, Yu, Jan, & Jan, 1997). Mutations of other residues in the PH, 70
such as Ser148 or Glu152 of mouse Kir3.2, also impair the K
+
selectivity (Chen et al., 71
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 11, 2021. ; https://doi.org/10.1101/2021.08.18.456735doi: bioRxiv preprint

4
2019; Yi, Lin, Jan, & Jan, 2001). Mutations located in transmembrane domains (TMs) 72
outside the SF also alter ion selectivity and residues located at the central cavity can 73
also influence the ion selectivity through interaction with the permeating ions (Bichet, 74
Grabe, Jan, & Jan, 2006; Bichet et al., 2004; Matamoros & Nichols, 2021; Yi et al., 75
2001). Structures of various types of K
+
channel have been identified by Cryo-EM 76
and X-ray crystallography (Long, Campbell, & Mackinnon, 2005; Miller & Long, 2012; 77
W. Wang & MacKinnon, 2017; Whorton & MacKinnon, 2011). However, the structural 78
changes induced by mutations at different regions of the channel which alter the ion 79
selectivity still remain to be elucidated. 80
Previous studies using homology modeling have predicted structural changes to 81
GIRK channels induced by disease associated mutations as follows: (A) mutations in 82
the SF (human Kir3.2 T152del or Kir3.2 G154S) impair the stabilization of K
+
83
interaction with the backbone of the SF (Masotti et al., 2015); (B) the L171R mutation 84
in the TM2 of human Kir3.2 limits the SF movement by the formation of a hydrogen 85
bond with Glu148 in the PH (Horvath et al., 2018), while the L168R mutation at the 86
corresponding position of human Kir3.4 is relevant to the interaction with the side 87
chain of Tyr of the GYG motif in the SF (Choi et al., 2011); (C) the T158A mutation in 88
the extracellular loop of human Kir3.4 weakens the stability of the structure by 89
eliminating the hydrogen bonds between the extracellular loop, PH, and TM1 (Choi et 90
al., 2011). However, no further experimental data have been provided to support 91
these hypotheses. 92
In the present study, we investigated the mechanisms underlying the abnormal ion 93
selectivity of these inherited mutations in Kir3.2 and Kir3.4 channels by introducing 94
mutations at the corresponding positions in mouse Kir3.2 or rat Kir3.4. Using 95
electrophysiological recordings in Xenopus oocytes, we demonstrate that most of 96
these mutations induce a conformational change which allows increased permeation 97
of Rb
+
or Cs
+
, while the G156S mutation of the Kir3.2 channel (corresponding to 98
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 11, 2021. ; https://doi.org/10.1101/2021.08.18.456735doi: bioRxiv preprint

5
human Kir3.2 G154S) induces a different conformational change which allows better 99
permeation of Li
+
or Na
+
over Rb
+
or Cs
+
. Applications of pore blockers of GIRK 100
channels increase the Li
+
-selectivity of Kir3.2 G156S but do not influence that of the 101
other mutants. Single-channel recordings of the G156S mutant in mouse fibroblasts 102
show that two types of ion conducting events, which respectively reflect K
+
current via 103
the SF pathway and Li
+
current via a novel ion permeation pathway, exist in the same 104
recording. Mutations of amino acid residues located in the PH behind the SF also 105
gave rise to the Li
+
-permeable pathway outside of the SF route. Our results reveal a 106
novel mechanism underlying the loss of K
+
-selectivity of Kir3.2 G156S that involves 107
formation of a novel ion permeation pathway in addition to the conventional SF route. 108
109
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted September 11, 2021. ; https://doi.org/10.1101/2021.08.18.456735doi: bioRxiv preprint