TWIK-Related K+ channels (TREK), including TREK-1 and TREK-2, belong to the TREK/TRAAK subclass of two-pore domain K+ (K2P) family. The important functions of transmembrane segment 4 (M4)-glycine hinge in TREK channel gating have been characterized, but the roles of M2-hinge (the equivalent residue of M4-hinge) remain unclear. Here, by characterizing the macroscopic currents, subcellular localization and gating properties of their M2-hinge mutants (G166A for TREK-1 and G196A for TREK-2), we investigated the functions of M2-hinge. G166A displayed decreased whole-cell currents, whereas no current was produced by G196A. Subcellular analysis indicated that both mutants were aggregated near the perinuclear region, and most of them were retented within the endoplasmic reticulum (ER). Next, to explore the roles of M2-hinge in the gating mechanism, we tested the responses of the related M2-hinge mutants to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH alteration (Delta pH(o)). TREK-1mut7-G166A displayed reduced sensitivity to 2-APB activation, but similar sensitivity to Delta pH(o), when compared with TREK-imut7. WT-Delta pCt, a TREK-2 tandom dimer, was used to assess the function of M2-hinge in the cis-type gating of TREK-2. The sensitivities of G196A-Delta pCt to both 2-APB and Delta pH(o) decreased compared with WT-Delta pCt. Taken together, our results reveal that the M2-hinge of TREK channels control their macroscopic current, subcellular localization and gating process. (C) 2017 Elsevier Inc. All rights reserved.