Single channel studies on human TREK 1 hTREK 1 channels to intracellular ischemia related factors

Abstract

TREK-1, a member of the two-pore domain family of potassium channels, majorly contributes to the maintenance of resting membrane potential of a cell and has been reported to respond to ischemic levels of intracellular lactate and acidic pH to provide neuroprotection. There are two N-terminal variants that arise due to Alternative splicing: the shorter variant having a shorter N-terminus than the full-length human TREK-1 (hTREK-1) which is widely expressed in the acute hypoxia sensitive regions of the adult brain like the cerebellum and hippocampus and is upregulated under ischemia. Previous whole-cell patch-clamp experiments on the shorter variant of hTREK1 have shown contradictory results to hypoxia- a condition attributed to ischemia, which has put the neuroprotective role of the hTREK-1 channel into question. Although these experiments were performed on the shorter hTREK-1, there have been no studies on the effect of hypoxia and other ischemia-related factors like lactate, pH, and hemin on the full-length hTREK-1 channel. In the present study, using single-channel inside-out patch-clamp electrophysiology on the full-length hTREK-1 expressed in HEK293 cells, we show that the extended N-terminus of the full-length hTREK-1 channel is required to sense hypoxia. The probability of opening of the full-length hTREK-1 channel reversibly increased on exposure to hypoxia. However, there was a decrease in the open probability of the shorter hTREK-1 channel under similar conditions suggesting that the N-terminus might be responsive to hypoxia or it might interact with the Cterminus of the protein or a sensor situated elsewhere in the channel. Due to the shift in glucose metabolism from aerobic to anaerobic mode upon ischemia, there is an increase in intracellular lactate that has been shown to be a potent modulator of the fulllength hTREK-1 channel. However, the modulatory effect of lactate and hypoxia on the fulllength hTREK-1 has not been reported...