TREK-2 expressed in mammalian cells exhibits small (similar to 52 pS) and large (similar to 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 (Delta 1-36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Delta 1-66) resulted in the appearance of only the large-conductance channel (similar to 220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Delta 1-44 and Delta 1-54 produced similar to 90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at similar to 54 kDa and similar to 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 similar to 60 kDa isoform and the small-conductance channel (similar to 52 pS). Mutants designed to produce translation from the second (M2L3) or third (M-3) initiation site produced the similar to 54 kDa isoform, and the large conductance channel (similar to 185-224 pS). M1L2L3, M2L3 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 M1L2L3 (similar to 0.02), M2L3 (similar to 0.02) and M-3 (similar to 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.
