The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltagedependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (

Mechanosensitive ion channels use mechanical energy to gate the dissipation of electrochemical gradients across cell membranes. This function is fundamental to physiological processes such as hearing and touch. In electrophysiological studies of ion channels in the plasma membrane of the yeast Saccharomyces cerevisiae, channels were observed that were activated by, and adapted to, stretching of the membrane. Adaptation of channel activity to mechanical stimuli was voltage-dependent. Because these mechanosensitive channels pass both cations and anions, they may play a role in turgor regulation in this walled organism.

Whether animal ion channels functioning as mechanosensors are directly activated by stretch force or indirectly by ligands produced by the stretch is a crucial question. TRPV4, a key molecular model, can be activated by hypotonicity, but the mechanism of activation is unclear. One model has this channel being activated by a downstream product of phospholipase A 2 , relegating mechanosensitivity to the enzymes or their regulators. We expressed rat TRPV4 in Xenopus oocytes and repeatedly examined >200 excised patches bathed in a simple buffer. We found that TRPV4 can be activated by tens of mm Hg pipette suctions with open probability rising with suction even in the presence of relevant enzyme inhibitors. Mechanosensitivity of TRPV4 provides the simplest explanation of its various forcerelated physiological roles, one of which is in the sensing of weight load during bone development. Gain-of-function mutants cause heritable skeletal dysplasias in human. We therefore examined the brachyolmia-causing R616Q gain-of-function channel and found increased whole-cell current densities compared with wild-type channels. Single-channel analysis revealed that R616Q channels maintain mechanosensitivity but have greater constitutive activity and no change in unitary conductance or rectification.In stark contrast with vision, smell, and most tastes, which are based on G-protein-coupled receptors, the mechanical senses (hearing, touch, balance, monitoring blood pressure or systemic osmolarity etc.) are poorly understood in molecular terms. Although prokaryotic mechanosensitive channels are clearly activated directly by membrane stretch force (6), how eukaryotic mechanosensitive channels are activated is still under debate. Models range from direct activation by stretches from membrane and/or cytoskeleton to indirect activation through stretch-produced ligands (7,8). The mammalian twopore K ϩ channels TREK1 and TAASK are activated by both membrane stretch and polyunsaturated fatty acids (PUFAs) 2 (9). There, cytoskeletal disruption actually increases mechanosensitivity, suggesting that the cytoskeleton does not transmit stretch force to these channels (10).Among the Ca 2ϩ -permeable transient receptor potential channels, yeast TRPY1 (11) and animal TRPV4 (transient receptor potential channel subtype V4) (12) have been studied extensively for their response to osmotic or mechanical stimuli. TRPY1 can be activated directly by suctions applied to excised membrane patches (11, 13). However, results from such a test from limited preliminary studies found in the TRPV4 literature are inconsistent and even contradictory (1, 2, 14), likely reflecting mechanical complexities of patches excised from animal cells and/or molecular heterogeneity.Rodent TRPV4 channels were first cloned repeatedly by following hypotonicity-induced Ca 2ϩ signals (1, 2). Rat trpv4 complements the mechano-and osmosensitivity defects of the osm-9 mutant worm (15). How osmotic force activates TRPV4 is unclear, however. Besides hypotonicity, this polymodal channel is a...

Ca 2؉ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca 2؉ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg ‫؍‬ 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca 2؉ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca 2؉ concentration and is apparently independent of its known Ca 2؉ activation, which we now propose to be an amplification mechanism (Ca 2؉ -induced Ca 2؉ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.

Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infection structures. Protoplasts from germ tubes of this fungus, when examined with patch-clamp electrodes, displayed the activities of a 600-picosiemen mechanosensitive ion channel. This channel passes a variety of cations, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This channel could transduce the membrane stress induced by the leaf topography into an influx of ions, including Ca2+, that may trigger differentiation.