Oxygen sensing by human recombinant K+ channels: assessment of the use of stable cell lines

Kemp, Paul J. ORCID: https://orcid.org/0000-0003-2773-973X, Iles, David and Peers, Chris 2004. Oxygen sensing by human recombinant K+ channels: assessment of the use of stable cell lines. Methods in Enzymology 381 , pp. 257-274. 10.1016/S0076-6879(04)81018-8

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Abstract

This chapter assesses the use of stable cell lines for oxygen sensing by human recombinant K+ channels. Central to the cardiorespiratory responses to reduced O2 availability of all chemosensory tissue is the rapid inhibition of ion channels by hypoxia. Acute hypoxic modulation of K+ channel activity is central to chemosensing in the carotid body, neuroepithelial body, and its immortalized cellular counterpart (H146cells). In this study, the impact that recombinant human K+ channel expression systems have on the understanding of acute O2 chemotransduction by native and model chemosensory tissues is discussed. Different aspects related to generating stable cell lines, including the choice of cell line, cell culture, choice of vector, ligation of cloned channels into pcDNA3.1-TOPO, transfection and selection, verification of expression by RT-PCR, verification of expression by immunocytochemistry, and functional screening using the patch-clamp technique are discussed. . Homomultimeric O2-sensitive human K+ channels stably expressed in HEK293 cells and alternative approaches to the study of heteromultimeric O2-sensitive human K+ channels are also described.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	Elsevier
ISSN:	0076-6879
Last Modified:	27 Oct 2022 08:47
URI:	https://orca.cardiff.ac.uk/id/eprint/63413

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