Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

SummaryThe relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of¯uorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a¯uorescent chimaera of the K -inward rectifying channel from Arabidopsis thaliana and the green¯uorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspeci®c labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were¯uorescently labelled. The identi®cation is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K -channel KAT1 from the PM via endocytic vesicles.

SummaryTraf®cking of K inward (K in ) rectifying channels was analyzed in guard cells of Vicia faba transfected with the K in recti®er from Arabidopsis thaliana KAT1 fused to the green¯uorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements con®rmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The K in recti®er current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active K in recti®er in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that K in recti®er remains in clusters during traf®cking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K channels preferentially in clusters during constitutive and pressure-driven exo-and endocytosis.

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K 1 channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.

The bacterial mechanosensitive channel of large conductance (MscL) is an ideal starting point for understanding the molecular basis of mechanosensation. However, current methods for the characterization of its mutants, patch clamp and bacterial growth analysis, are difficult and time consuming, so a higher throughput method for screening mutants is desired. We have attempted to develop a fluorescence assay for detecting MscL activity in synthetic vesicles. The assay involved the separation of two solutionsone inside and one outside the vesicles-that are separately nonfluorescent but fluorescent when mixed. It was hoped that MscL activity due to downshock of the vesicles would bring about mixing of the solutions, producing fluorescence. The development of the assay required the optimization of several variables: the method for producing a uniform vesicle population containing MscL, the fluorescence system, and the lipid and protein composition of the vesicles. However, no MscL activity was ever detected even after optimization, so the assay was not fully developed. The probable cause of the failure was the inability of current techniques to produce a sufficiently uniform vesicle population.