Abstract

Two-pore domain potassium (K(2P)) 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 K(2P) 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 K(2P) channels possess consensus N-linked glycosylation sites, and here we demonstrate that the conserved putative N-glycosylation site in K(2P)3.1 and K(2P)9.1 is a glycan acceptor site. Patch clamp analysis revealed that disruption of channel glycosylation reduced K(2P)3.1 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 of an effect on K(2P)9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. Because nonglycosylated channels appear to pass through the secretory pathway in a manner comparable with glycosylated channels, the evidence presented here suggests that the decreased number of nonglycosylated K(2P)3.1 channels on the cell surface may be due to their decreased stability.

Highlights

  • N-Glycosylation regulates the function of many membrane proteins

  • Summary of K2P constructs used in this study Channels were tagged with eGFP fused to the N terminus, with or without an HA epitope tag in the external loop of the second pore-forming domain

  • Glycosylation of K2P Channels—Protein sequences of human, mouse, and rat K2P channels were analyzed for the presence of N-glycosylation consensus sites using the NetNGlyc 1.0 server [21]

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Summary

Introduction

N-Glycosylation regulates the function of many membrane proteins. Results: K2P3.1 and K2P9.1 possess functional glycosylation sites, and a lack of glycosylation results in fewer channels on the cell surface. Conclusions: N-Linked glycosylation has a critical role in K2P3.1 and a modulatory role in K2P9.1 cell surface expression. 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, influencing cellular membrane potential and activity. Regulation of these channels is of critical importance to cellular function. Disruption of N-linked glycosylation has less of an effect on K2P9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. Because nonglycosylated channels appear to pass through the secretory pathway in a manner comparable with glycosylated channels, the evidence presented here suggests that the decreased number of nonglycosylated K2P3.1 channels on the cell surface may be due to their decreased stability

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