Membrane Mechanics, Osmotic Stress, and the Yeast Stretch-Activated TRP

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Although vision, olfaction and taste are understood, at the level of molecules, we do not understand mechanical senses such as touch, hearing, blood pressure, osmotic balance. Even when ion channels of the TRP superfamily are suspected to transduce mechanical force into ion flux, progress has been slow with animal preparations because of anatomical complexity, shortage of relevant materials, and cumbersome genetics. We will complement the animal work by combining the prowess of yeast molecular genetics and the resolution of patch clamp on the study of a TRP channel in yeast. The yeast TRP channel, Yvc1p, instantly opens when cells are confronted with hyperosmolarity (= dehydration) to release Ca2+ from vacuole to cytoplasm. Under patch clamp, Yvc1p channels are directly activated by applied membrane stretch force. Channels from yvcl mutants selected after a random mutagenesis are found to retain their mechanosensitivity but are unstable in their closed Or open conformations. These mutations turn out to involve aromatic residues, known to often in locations that parallel the lipid bilayer's interfacial level. The interface centers between the charged lipid head and the hydrophobic tail of the lipid bilayer, a point where surface tension is the highest. It appears that the aromatic residues of the channel protein anchor the protein at this level to stabilize its normal conformations. We hypothesize that added stretch force perturbs the force distribution within the bilayer. This perturbation eventually reaches the channel "gate" (the ion pathway occlusion) through the aromatic anchors. Consistent with the hypothesis, exogenously added aromatic compounds (tryptophan, indole, parabens, etc) have recently been found to activate Yvc1p in vivo and under patch clamp. We will continue to generate mutants with amino-acid substitutions or deletions to define the mechanism of and the domains required for force transmission. Aromatic and amphipathic compounds that perturb the interfaces of the bilayer will be systematically examined to test the interface-tension hypothesis.