Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

B Heitz; R Branstrom; W Yang; Y Huang; T Moede; I Leibiger; B Leibiger; L Chen; J Yu; S Yang; O Larsson; S Saavedra; Per-Olof Berggren; C Aspinwall

doi:10.1038/s41598-021-00988-y

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

B Heitz, R Branstrom, W Yang, Y Huang, T Moede, I Leibiger, B Leibiger, L Chen, J Yu, S Yang, O Larsson, S Saavedra, Per-Olof Berggren, C Aspinwall

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Abstract

ATP-sensitive K+ (K_ATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type K_ATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Original language	English
Article number	21539 (2021)
Number of pages	11
Journal	Scientific Reports
Volume	11
Issue number	1
Early online date	2 Nov 2021
DOIs	https://doi.org/10.1038/s41598-021-00988-y
Publication status	Published (in print/issue) - Dec 2021

Bibliographical note

Funding Information:
This work was supported by National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Nos. EB007047 and EB022297 and the National Science Foundation (0548167, 2003297). Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and Karolinska Institutet; Swedish Society of Medicine (Bengt Ihre grant); the Swedish Research Council; the Novo Nordisk Foundation; the Swedish Diabetes Association; the Family Erling-Persson Foundation; the Skandia Insurance Company Ltd; Strategic Research Program in Diabetes at Karolinska Institutet; the Berth von Kantzow’s Foundation; the Knut and Alice Wallenberg Foundation; Funds of Karolinska Institutet; Diabetes and Wellness Foundation; the Stichting af Jochnick Foundation; ERC-2018-AdG 834860 EYELETS.

Publisher Copyright:
© 2021, The Author(s).

© 2021. The Author(s).

Keywords

Adenosine Diphosphate/metabolism
Adenosine Triphosphate/metabolism
Animals
Cell Membrane/metabolism
HEK293 Cells
Humans
Ion Channel Gating
Oocytes/cytology
Potassium Channels, Inwardly Rectifying/genetics
Sulfonylurea Receptors/genetics
Xenopus laevis

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Heitz, B., Branstrom, R., Yang, W., Huang, Y., Moede, T., Leibiger, I., Leibiger, B., Chen, L., Yu, J., Yang, S., Larsson, O., Saavedra, S., Berggren, P.-O., & Aspinwall, C. (2021). Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels. Scientific Reports, 11(1), Article 21539 (2021). https://doi.org/10.1038/s41598-021-00988-y

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abstract = "ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.",

keywords = "Adenosine Diphosphate/metabolism, Adenosine Triphosphate/metabolism, Animals, Cell Membrane/metabolism, HEK293 Cells, Humans, Ion Channel Gating, Oocytes/cytology, Potassium Channels, Inwardly Rectifying/genetics, Sulfonylurea Receptors/genetics, Xenopus laevis",

author = "B Heitz and R Branstrom and W Yang and Y Huang and T Moede and I Leibiger and B Leibiger and L Chen and J Yu and S Yang and O Larsson and S Saavedra and Per-Olof Berggren and C Aspinwall",

note = "Funding Information: This work was supported by National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Nos. EB007047 and EB022297 and the National Science Foundation (0548167, 2003297). Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and Karolinska Institutet; Swedish Society of Medicine (Bengt Ihre grant); the Swedish Research Council; the Novo Nordisk Foundation; the Swedish Diabetes Association; the Family Erling-Persson Foundation; the Skandia Insurance Company Ltd; Strategic Research Program in Diabetes at Karolinska Institutet; the Berth von Kantzow{\textquoteright}s Foundation; the Knut and Alice Wallenberg Foundation; Funds of Karolinska Institutet; Diabetes and Wellness Foundation; the Stichting af Jochnick Foundation; ERC-2018-AdG 834860 EYELETS. Publisher Copyright: {\textcopyright} 2021, The Author(s). {\textcopyright} 2021. The Author(s).",

year = "2021",

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doi = "10.1038/s41598-021-00988-y",

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T1 - Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

AU - Heitz, B

AU - Branstrom, R

AU - Yang, W

AU - Huang, Y

AU - Moede, T

AU - Leibiger, I

AU - Leibiger, B

AU - Chen, L

AU - Yu, J

AU - Yang, S

AU - Larsson, O

AU - Saavedra, S

AU - Berggren, Per-Olof

AU - Aspinwall, C

N1 - Funding Information: This work was supported by National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Nos. EB007047 and EB022297 and the National Science Foundation (0548167, 2003297). Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and Karolinska Institutet; Swedish Society of Medicine (Bengt Ihre grant); the Swedish Research Council; the Novo Nordisk Foundation; the Swedish Diabetes Association; the Family Erling-Persson Foundation; the Skandia Insurance Company Ltd; Strategic Research Program in Diabetes at Karolinska Institutet; the Berth von Kantzow’s Foundation; the Knut and Alice Wallenberg Foundation; Funds of Karolinska Institutet; Diabetes and Wellness Foundation; the Stichting af Jochnick Foundation; ERC-2018-AdG 834860 EYELETS. Publisher Copyright: © 2021, The Author(s). © 2021. The Author(s).

PY - 2021/12

Y1 - 2021/12

N2 - ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

AB - ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

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KW - Adenosine Triphosphate/metabolism

KW - Animals

KW - Cell Membrane/metabolism

KW - HEK293 Cells

KW - Humans

KW - Ion Channel Gating

KW - Oocytes/cytology

KW - Potassium Channels, Inwardly Rectifying/genetics

KW - Sulfonylurea Receptors/genetics

KW - Xenopus laevis

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SN - 2045-2322

VL - 11

JO - Scientific Reports

JF - Scientific Reports

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M1 - 21539 (2021)

ER -

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

Abstract

Bibliographical note

Keywords

UN SDGs

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