Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
TREK-2 expressed in mammalian cells exhibits small ( approximately 52 pS) and large ( approximately 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 (Delta1-36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Delta1-66) resulted in the appearance of only the large-conductance channel ( approximately 220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Delta1-44 and Delta1-54 produced approximately 90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at approximately 54 kDa and approximately 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 approximately 60 kDa isoform and the small-conductance channel ( approximately 52 pS). Mutants designed to produce translation from the second (M(2)L(3)) or third (M(3)) initiation site produced the approximately 54 kDa isoform, and the large conductance channel ( approximately 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) ( approximately 0.02), M(2)L(3) ( approximately 0.02) and M(3) ( approximately 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.
Department of Physiology & Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA. donghee.kim@rosalindfranklin.edu
Dina
D
Simkin
Eric J
EJ
Cavanaugh
Donghee
D
Kim
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011312:Pressure; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014162:Transfection
C503444:Kcnk10 protein, rat; C503434:Kcnk4 protein, rat; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D020033:Protein Isoforms; C103742:potassium channel protein TREK-1; D016718:Arachidonic Acid; D012964:Sodium; D011188:Potassium