Control of the single channel conductance of K 2P 10.1 (TREK‐2) by the amino‐terminus: role of alternative translation initiation

• Published in: The Journal of physiology Vol. 586; no. 23; pp. 5651 - 5663
• Main Authors: Simkin, Dina, Cavanaugh, Eric J., Kim, Donghee
• Format: Journal Article
• Language: English
• Published: 01.12.2008
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2008.161927

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 (Δ1–36) of the N‐terminus had no effect. However, deletion of most of the N‐terminus (Δ1–66) 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 Δ1–44 and Δ1–54 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 M 1 M 2 M 3 ) within the N‐terminus. Mutation of the second and third initiation sites from Met to Leu (M 1 L 2 L 3 ) produced only the ∼60 kDa isoform and the small‐conductance channel (∼52 pS). Mutants designed to produce translation from the second (M 2 L 3 ) or third (M 3 ) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M 1 L 2 L 3 , M 2 L 3 and M 3 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 M 1 L 2 L 3 (∼0.02), M 2 L 3 (∼0.02) and M 3 (∼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.