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J Physiol Volume 583, Number 1, 57-69, August 15, 2007 DOI: 10.1113/jphysiol.2007.136150
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CELLULAR

Properties of single two-pore domain TREK-2 channels expressed in mammalian cells

 Dawon Kang1,2, Changyong Choe1,3, Eric Cavanaugh1 and Donghee Kim1

1 Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064 USA
2 Medical Research Center for Neural Dysfunction and Department of Physiology, Gyeongsang National University College of Medicine, Jinju 660-751, Korea
3 National Livestock Research Institute, Namwon 590-832, Korea

TREK-2 (K2P10.1), a member of the two-pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK-2 exhibits small (TREK-2S) and large (TREK-2L) conductance phenotypes when expressed in mammalian cell lines (COS-7, HEK293, HeLa) and in Xenopus oocytes. TREK-2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK-2L phenotype shows a full open state (202 pS) with several short-lived sub-conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (–40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK-2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK-2S and TREK-2L, and caused partial switching of conductance levels from those of TREK-2S and TREK-2L to more intermediate values. Under this condition, TREK-2 exhibited six conducting levels and one closed level. TREK-2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK-2S and TREK-2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK-2 in Mg2+-free solution. Phenotypes resembling TREK-2S and TREK-2L were also observed in cerebellar granule neurons that express TREK-2 mRNA. These results show that TREK-2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability.

(Received 9 May 2007; accepted after revision 30 May 2007; first published online 31 May 2007)
Corresponding author D. Kim: Department of Physiology & Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA. Email: donghee.kim{at}rosalindfranklin.edu






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