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

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 sequenc...

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Autores principales:	Benjamin A. Heitz, Robert Bränström, Wei Yang, Yiding Huang, Tilo Moede, Ingo B. Leibiger, Barbara Leibiger, Liu Qi Chen, Jia Yu, Shao-Nian Yang, Olof Larsson, S. Scott Saavedra, Per-Olof Berggren, Craig A. Aspinwall
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/87b22a90ce764dbc9e3214f3647c135f
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spelling	oai:doaj.org-article:87b22a90ce764dbc9e3214f3647c135f2021-11-08T10:47:29ZExpression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels10.1038/s41598-021-00988-y2045-2322https://doaj.org/article/87b22a90ce764dbc9e3214f3647c135f2021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-00988-yhttps://doaj.org/toc/2045-2322Abstract 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.Benjamin A. HeitzRobert BränströmWei YangYiding HuangTilo MoedeIngo B. LeibigerBarbara LeibigerLiu Qi ChenJia YuShao-Nian YangOlof LarssonS. Scott SaavedraPer-Olof BerggrenCraig A. AspinwallNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Benjamin A. Heitz Robert Bränström Wei Yang Yiding Huang Tilo Moede Ingo B. Leibiger Barbara Leibiger Liu Qi Chen Jia Yu Shao-Nian Yang Olof Larsson S. Scott Saavedra Per-Olof Berggren Craig A. Aspinwall Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
description	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.
format	article
author	Benjamin A. Heitz Robert Bränström Wei Yang Yiding Huang Tilo Moede Ingo B. Leibiger Barbara Leibiger Liu Qi Chen Jia Yu Shao-Nian Yang Olof Larsson S. Scott Saavedra Per-Olof Berggren Craig A. Aspinwall
author_facet	Benjamin A. Heitz Robert Bränström Wei Yang Yiding Huang Tilo Moede Ingo B. Leibiger Barbara Leibiger Liu Qi Chen Jia Yu Shao-Nian Yang Olof Larsson S. Scott Saavedra Per-Olof Berggren Craig A. Aspinwall
author_sort	Benjamin A. Heitz
title	Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
title_short	Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
title_full	Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
title_fullStr	Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
title_full_unstemmed	Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels
title_sort	expression of truncated kir6.2 promotes insertion of functionally inverted atp-sensitive k+ channels
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/87b22a90ce764dbc9e3214f3647c135f
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Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

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