Sensing the pressure

Mechano-sensitive ion channels convert external mechanical forces into electrical and chemical signals in the cell. In the case of the TREK-1 channel, it is opened by membrane stretch and cell swelling, which results in outward rectifying potassium currents and shaping of the action potential. Reporting in the EMBO Journal, Chemin and colleagues found that phospholipids are important in this mechanical gating process.

An artificial increase in the cytosolic concentration of phosphatidylinositol 4,5-bisphosphate (PIP₂) — a component of membrane phospholipids — stimulates the activity of endogenous TREK-1 in cultured striatal neurons. This elevation in channel activity is also seen when the cytosol is acidified, and the effects of both PIP₂ and low intracellular pH can be inhibited by the presence of positively charged intracellular molecules such as polylysine. The inhibitory effect of polylysine molecules is thought to be due to their interaction with the negative charges of membrane phospholipids, which prevents them from interacting electrostatically with specific positively charged segments in the channel protein.

The authors also show that PIP₂ and acid can regulate the gating of TREK-1 in a cooperative manner: when TREK-1 is challenged with acid after addition of PIP₂, it becomes 'locked' in a highly active form even after the cytosolic pH is returned to pH 7.2. The activity of the locked TREK-1 channel is high even at atmospheric pressure, and pressure stimulation produces only a modest further activating effect. TREK-1 activation becomes more resistant to membrane stretch in the presence of polylysine, which indicates that the cationic molecule interferes with the pressure-sensing process.

Which segment of the channel is responsible for PIP₂ stimulation? The authors generated truncated, chimaeric and point mutations in TREK-1, and analysed the channel activity in cells that overexpressed the mutant proteins. They found that a protein segment of the cytosolic carboxy-terminal domain of TREK-1 is crucial for PIP₂ stimulation. This region contains several positively charged resides that seem to be attractive candidates for binding to phospholipids. A systematic substitution of the positively charged amino acids by alanines, which are neutral, progressively leads to reduced TREK-1 stimulation by PIP₂. The authors conclude that the cationic cluster is important for stimulation of TREK-1 by phospholipids.

Chemin and colleagues then reasoned that if this positively charged region of TREK-1 is to interact with phospholipids, it must be localized near the plasma membrane. To show this, they used a complementation yeast system, in which a mutant strain fails to grow at the non-permissive temperature of 37 °C unless it is rescued by the exogenously expressed amino-terminal fragment of hSOS, which lacks the membrane-targeting activity. As this fragment of hSOS is only functional when it gets to the plasma membrane, mutant yeast cells are not rescued unless hSOS is fused to another protein or protein segment that interacts with the plasma membrane. The authors show that the cytosolic cationic cluster of TREK-1 brought the hSOS fragment to the plasma membrane and allowed cell growth at 37 °C.

These findings have shed new light on the gating mechanism of the TREK channels, and might also have more general implications for other types of mechanically activated ion channel, as Hirono and colleagues recently suggested for the hair cell channel.