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¹ Department of Physiology and Biophysics, and the Interdisciplinary Neuroscience Program, Rosalind Franklin University of Medicine and Science, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA

TREK-2 expressed in mammalian cells exhibits small (52 pS) and large (220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (1–36) of the N-terminus had no effect. However, deletion of most of the N-terminus (1–66) resulted in the appearance of only the large-conductance channel (220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants 1–44 and 1–54 produced 90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at 54 kDa and 60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M₁M₂M₃) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M₁L₂L₃) produced only the 60 kDa isoform and the small-conductance channel (52 pS). Mutants designed to produce translation from the second (M₂L₃) or third (M₃) initiation site produced the 54 kDa isoform, and the large conductance channel (185–224 pS). M₁L₂L₃, M₂L₃ and M₃ were relatively selectively permeable to K⁺, as judged by the 51–55 mV shifts in reversal potential following a 10-fold change in [K⁺]_o. P_Na/P_K values were also similar for M₁L₂L₃ (0.02), M₂L₃ (0.02) and M₃ (0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance.

(Received 19 August 2008; accepted after revision 7 October 2008; first published online 9 October 2008)
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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