IntroductionPotassium (K+) channels play a crucial roles in the regulation of the resting membrane potential and excitability of various cells. The mammalian two‐pore domain K+ channels are responsible for background or leak K+ currents, one of which is TREK‐1 (TWIK‐related K+ channel). The current of TREK‐1 channel appears to be regulated by intracellular transport, in which the cytosolic C‐terminus has been shown to play a important role in cell surface expression of the channel. An interesting feature of the TREK‐1 channel is the run‐up: i.e. the current through TREK‐1 channels spontaneously increases within several minutes in the whole‐cell patch‐clamp mode. Since C‐terminal domain is required for the surface localization of the TREK‐1 channel, the run‐up might be regulated by transport to the plasma membrane.ObjectivesThe present study aimed to investigate the involvement of intracellular transport in the run‐up.MethodsWe stably transduced TREK‐1c or its fusion protein with mCherry to the 293T cells and measured whole‐cell currents with patch‐clamp techniques. Cell surface localization was analyzed with confocal microscopic analysis and protein biotinylation.ResultsInhibitors of membrane protein transport, N‐methylmaleimide (NEM) and brefeldin‐A, inhibited the run‐up, while an endocytosis inhibitor, pitstop2, facilitated the run‐up. Correspondingly, in cells stably expressing the mCherry‐TREK‐1c fusion protein, NEM decreased the surface localization of mCherry‐TREK‐1c, whereas pitstop2 increased it. The run‐up was inhibited by the intracellular application of a peptide of the C‐terminal fragment, TREK335‐360, corresponding to the interaction site with microtubule associated protein 2 (Mtap2). The extracellular application of an Ezrin inhibitor (NSC668394) also suppressed the surface localization and the run‐up. The co‐application of these inhibitors abolished the TREK‐1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK‐1c channels and run‐up.ConclusionThese results indicate that intracellular protein transport is involved in the run‐up of TREK‐1c currents.COI: NOKey words: K+ channel, TREK‐1, intracellular transport, microtubule‐associated protein 2, ezrinSupport or Funding InformationThis study was supported by KAKENHI from the JSPS, the SICP from the JST, the Heiwa Nakajima Foundation, the Ryobi Memorial Foundation (to MO), the Mishima Memorial Foundation, the Rotary Yoneyama Memorial Foundation (to NA), and the Fujii Memorial Foundation (to AJ).