The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2

TREK-2 (TWIK-related K+ channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K+. Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba2+ inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K+ or mutation of S4 K+ binding site, which suggests that the inhibitory mechanism of Ba2+ is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba2+ and alteration of extracellular pH (pH(o)), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pH(o), which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels.