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

TLDR: 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.

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.
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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