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N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1

N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1
N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1
Two-pore domain potassium (K2P) channels play fundamental roles in cellular processes by enabling a constitutive leak of potassium from cells in which they are expressed, thus influencing cellular membrane potential and activity. Hence, regulation of these channels is of critical importance to cellular function. A key regulatory mechanism of K2P channels is the control of their cell surface expression. Membrane protein delivery to and retrieval from the cell surface is controlled by their passage through the secretory and endocytic pathways and post-translational modifications regulate their progression through these pathways. All but one of the K2P channels possess consensus N-linked glycosylation sites and here we demonstrate that the conserved putative N-glycosylation site in K2P3.1 and K2P9.1 is a glycan acceptor site. Patch-clamp analysis revealed that disruption of channel glycosylation reduced K2P3.1 current, and flow cytometry was instrumental in attributing this to a decreased number of channels on the cell surface. Similar findings were observed when cells were cultured in reduced glucose concentrations. Disruption of N-linked glycosylation has less effect on K2P9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. As non-glycosylated channels appear to pass through the secretory pathway in a manner comparable to glycosylated channels, evidence presented here suggests that the decreased number of non-glycosylated K2P3.1 channels on the cell surface may be due to their decreased stability.
K2P channel, TASK, K+ channel, N-linked glycosylation, membrane targeting, trafficking, surface expression
0021-9258
3251-3264
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Williams, Sarah
f98f47db-b1d6-42c2-b0eb-7c0cb9a981d0
Roncoroni, Laura
463bc93f-5a84-46fc-a6ff-ef2caa926cba
Lowry, Eleanor
701f9237-9dab-4c28-b9a5-effb8d3905d9
Johnson, Daniel
929aff41-fc03-40b8-a098-72c3ccda4b37
O'Kelly, Ita
e640f28a-42f0-48a6-9ce2-cb5a85d08c66
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Williams, Sarah
f98f47db-b1d6-42c2-b0eb-7c0cb9a981d0
Roncoroni, Laura
463bc93f-5a84-46fc-a6ff-ef2caa926cba
Lowry, Eleanor
701f9237-9dab-4c28-b9a5-effb8d3905d9
Johnson, Daniel
929aff41-fc03-40b8-a098-72c3ccda4b37
O'Kelly, Ita
e640f28a-42f0-48a6-9ce2-cb5a85d08c66

Mant, Alexandra, Williams, Sarah, Roncoroni, Laura, Lowry, Eleanor, Johnson, Daniel and O'Kelly, Ita (2013) N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1. The Journal of Biological Chemistry, 288 (5), 3251-3264. (doi:10.1074/jbc.M112.405167). (PMID:23250752)

Record type: Article

Abstract

Two-pore domain potassium (K2P) channels play fundamental roles in cellular processes by enabling a constitutive leak of potassium from cells in which they are expressed, thus influencing cellular membrane potential and activity. Hence, regulation of these channels is of critical importance to cellular function. A key regulatory mechanism of K2P channels is the control of their cell surface expression. Membrane protein delivery to and retrieval from the cell surface is controlled by their passage through the secretory and endocytic pathways and post-translational modifications regulate their progression through these pathways. All but one of the K2P channels possess consensus N-linked glycosylation sites and here we demonstrate that the conserved putative N-glycosylation site in K2P3.1 and K2P9.1 is a glycan acceptor site. Patch-clamp analysis revealed that disruption of channel glycosylation reduced K2P3.1 current, and flow cytometry was instrumental in attributing this to a decreased number of channels on the cell surface. Similar findings were observed when cells were cultured in reduced glucose concentrations. Disruption of N-linked glycosylation has less effect on K2P9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. As non-glycosylated channels appear to pass through the secretory pathway in a manner comparable to glycosylated channels, evidence presented here suggests that the decreased number of non-glycosylated K2P3.1 channels on the cell surface may be due to their decreased stability.

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Mant et al jbc M112 405167 full.pdf - Accepted Manuscript
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e-pub ahead of print date: 18 December 2012
Published date: February 2013
Keywords: K2P channel, TASK, K+ channel, N-linked glycosylation, membrane targeting, trafficking, surface expression
Organisations: Human Development & Health

Identifiers

Local EPrints ID: 346494
URI: http://eprints.soton.ac.uk/id/eprint/346494
ISSN: 0021-9258
PURE UUID: d0f6cb62-45b8-4928-9d24-f53658c116df
ORCID for Alexandra Mant: ORCID iD orcid.org/0000-0001-7169-209X

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Date deposited: 04 Jan 2013 13:36
Last modified: 27 Oct 2023 01:45

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Contributors

Author: Alexandra Mant ORCID iD
Author: Sarah Williams
Author: Laura Roncoroni
Author: Eleanor Lowry
Author: Daniel Johnson
Author: Ita O'Kelly

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