Construction Of Functional, Concatenated Dimers Of Two-pore-domain Potassium Channel TREK-1 And TREK-2

Objective: To explore the feasibility of adding a flexible linker between Two-pore-domain potassium channel TREK-1 (TWIK related K+ channel 1) and TREK-2(TWIK related K+ channel 2) monomers respectively to construct a tandem-linked dimer.Methods: PCR was used to add a flexible linker between the two TREK-1 monomers.The cRNA obtained from in vitro transcription using the above vector was injected into Xenopus oocytes. After 24-48 hours, currents were recorded from these oocytes using two-electrode voltage clamp. The effects of extracellular Ba2+ and pH on TdTREK-1 were observed and compared with those of native dimeric TREK-1. To construct the pGH19-TREK-2/TREK-2(WT-WT) concatenated dimer used for the methods mentioned above. The effects of 2-APB and pH on WT-WT were observed and compared with those of native dimeric TREK-2. Western blotting was used to detect protein expression of TREK-2 and WT-WT.Results: The tandem-linked dimeric TdTREK-1 was highly expressed in Xenopus oocytes. The currents through these channels were inhibited by extracellular Ba2+ and acidification. Furthermore, the responsiveness of the concatenated dimmers to these extracellular stimuli was similar to those of native dimers. The current and pharmacological properties of the WT-WT was similar to those of native dimers.Conclusion: Adding a flexible linker between the two monomers to construct the tandem-linked dimer does not affect the expression and properties of TREK-1 and TREK-2,suggesting that the method be feasible. Such method will allow the manipulation of a single subunit, which provides a further basis for studying the structure and function of TREK-1 and TREK-2.