Mechanotransduction Pathway For The Two-pore Domain K~+ Channel TREK-1

Mechanosensitive(MS)ion channels act as a molecular switch transducing mechanical stimuli into chemical or electrical signals and play an important role in mechanotransduction of many physiological processes,such as cardiovascular regulation,osmoregulation,hearing,balance,pain and touch.TREK-1,a member of two-pore domain K+ channel family,is the first mammalian mechanosensitive channel identified at the molecular level.TREK-1 produces'background' currents that stabilize the resting membrane potential and have a critical role in control or modulation of cell excitability.TREK-1 is physiologically involved in epilepsy,anesthesia,depression,pain,neuroprotection of ischemia.However,the gating mechanism of TREK-1 in response to mechanical stimulation is still largely unknown.In this study,we investigated the mechanical response pathway in TREK-1.Electrophysiological results showed that deletion of the C-terminal region(?Ct/TREK-1)completely abolished mechanosensitivity of TREK-1.However,the C-terminal of TREK-1 conferred mechanosensitivity to the non-mechanosensitive channel TWIK-1 of TWIK-1(1-264)/CtTREK-1 chimera.Meanwhile,C-terminal of TWIK-1 couldn't restore the mechanosensitivity of ? Ct/TREK-1.Those results confirmed the vital role of C-terminal in the mechanotransduction of TREK-1.Furthermore,we found that substitutions of 1299 caused significant reduction in the mechanosensitivity,suggesting that 1299 is a key point in C-terminal domain.We next address the question of how does the C-terminal transfer the mechanical information to the pore domain to activate the channel.Combining the sequence alignment and site-directed mutagenesis,we found that the glycine hinge of TM4(G281),which was conserved in this domain,was involved in the mechanotransduction.Large side chain substitutions at position 281(G281V,G281L,G281C)decreased the mechanical activation of TREK-1.We found that L249 at the end of pore-helix 2(PH2)and near the selectivity filter(SF),which is spatially in close proximity with G281,also participated in mechanotransduction.In contract to the effect of G281,small side chain substitutions at position of 249(L249G,L249C)produced a significant increase in mechanosensitivity.These results suggest that the interaction between G281 and L249 modulates the mechanosensation of TREK-1.Based on the analysis of crystal structures of homologous TREK-2,we found that the glycine hinge of TM2(G166)and 1140 in PHI are close to each other in the spatial conformation.Electrophysiological analysis indicated that they have the similar effect to that of G281 and L249,which both are involved in the mechanotransduction pathway of TREK-1.In summary,we proposed a mechanical response model of TREK-1.In this model,C-terminal transfers the mechanical stimulation to TM4 through 1299;subsequently G281 responds to the mechanical stimuli and leads to the conformation changes of TM4;mechanical information then transmits to PH2 and SF via the G281-L249 interaction,as well as to TM2 and PHI via G166-1140 interaction,and ultimately activates the TREK-1 channel.