Elucidation of the Interaction between Flavan-3-ols and Bovine Serum Albumin and Its Effect on Their In-Vitro Cytotoxicity.

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2; tetramer) > procyanidin C1 (C1; trimer) ≈ procyanidin B2 (B2, dimer) > (-)epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay. FLs exerted cytotoxicity at 1000 µg/mL in F11 cell culture with fetal bovine serum containing BSA. In culture containing serum-free medium, FLs exhibited significant cell proliferation at 10-4 µg/mL and cytotoxicity was observed at concentrations greater than 10 µg/mL. Results of this study suggest that interactions between polyphenols and BSA should be taken into account when evaluating procyanidin in an in vitro cell culture system.

Temperature-dependent augmentation of ciliary motility by the TRP2 channel in Chlamydomonas reinhardtii.

Temperature is a critical factor for living organisms. Many microorganisms migrate toward preferable temperatures, and this behavior is called thermotaxis. In this study, the molecular and physiological bases for thermotaxis are examined in Chlamydomonas reinhardtii. A mutant with knockout of a transient receptor potential (TRP) channel, trp2-3, showed defective thermotaxis. The swimming velocity and ciliary beat frequency of wild-type Chlamydomonas increase with temperature; however, this temperature-dependent enhancement of motility was almost absent in the trp2-3 mutant. Wild-type Chlamydomonas showed negative thermotaxis, but mutants deficient in the outer or inner dynein arm showed positive thermotaxis and a defect in temperature-dependent increase in swimming velocity, suggesting involvement of both dynein arms in thermotaxis.

Electrophysiological characterization of the mechanosensitive channel MscCG in Corynebacterium glutamicum.

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.

Mechanoresponses mediated by the TRP11 channel in cilia of Chlamydomonas reinhardtii.

Cilia are organelles involved in motility and sensory transduction, but how these two functions coexist has not been elucidated in depth. Here, the involvement of the ciliary transient receptor potential (TRP) channel TRP11 in mechanoresponses is studied in Chlamydomonas reinhardtii using a TRP11-knockout mutant. The mutant has defects in the conversion of the bending mode of the cilium from forward to reverse when tapped with a glass rod, the detachment of cilia when shear is applied, the increase in ciliary beat frequency upon application of mechanical agitation by vortex mixing, and the initiation of gliding while both cilia are attached in opposite directions to a glass surface. These observations indicate that TRP11 can perceive mechanical stimuli with distinct intensities and durations and induce various types of ciliary responses.

A gain-of-function mutation in gating of Corynebacterium glutamicum NCgl1221 causes constitutive glutamate secretion.

The A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion in Corynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

Gating-associated conformational changes in the mechanosensitive channel MscL.

Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3-4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patch-clamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, Ca(2+) influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically cross-linked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation.

MCAs in Arabidopsis are Ca2+-permeable mechanosensitive channels inherently sensitive to membrane tension.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca2+-permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca2+ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension.

The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.

Photo-induced behavioral responses (photobehaviors) are crucial to the survival of motile phototrophic organisms in changing light conditions. Volvocine green algae are excellent model organisms for studying the regulatory mechanisms of photobehavior. We recently reported that unicellular Chlamydomonas reinhardtii and multicellular Volvox rousseletii exhibit similar photobehaviors, such as phototactic and photoshock responses, via different ciliary regulations. To clarify how the regulatory systems have changed during the evolution of multicellularity, we investigated the photobehaviors of four-celled Tetrabaena socialis. Surprisingly, unlike C. reinhardtii and V. rousseletii, T. socialis did not exhibit immediate photobehaviors after light illumination. Electrophysiological analysis revealed that the T. socialis eyespot does not function as a photoreceptor. Instead, T. socialis exhibited slow accumulation toward the light source in a photosynthesis-dependent manner. Our assessment of photosynthetic activities showed that T. socialis chloroplasts possess higher photoprotection abilities against strong light than C. reinhardtii. These data suggest that C. reinhardtii and T. socialis employ different strategies to avoid high-light stress (moving away rapidly and gaining photoprotection, respectively) despite their close phylogenetic relationship.

Responses to transient receptor potential (TRP) channel agonists in Chlamydomonas reinhardtii.

Pungent substances, such as capsaicin and gingerol, activate the transient receptor potential (TRP)-V1 channel and affect the feeding behaviors of animals. To gain insight into how living organisms have acquired a sense for pungent substances, we explored the response to TRP agonists in a protist, Chlamydomonas reinhardtii When capsaicin or gingerol was applied to wild-type cells, they became immotile, with flagella detaching from the cell body. The degree of deflagellation was nearly halved in a mutant defective in the TRP channel ADF1. Deflagellation in the adf1 mutant was inhibited further by Ruthenium Red, indicating ADF1 and another TRP channel are involved in the deflagellation response. The response to capsaicin and gingerol was not inhibited by TRPV1-specific blockers such as 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) and capsazepine. When capsaicin or gingerol was applied to wild-type cells in the presence of Ruthenium Red, a large proportion lost motility while flagella remained attached, suggesting that flagella stop contributing to motility, at least in part, through a TRP-channel-independent pathway. These results indicate that pungent compounds such as capsaicin and gingerol induce loss of flagellar motility and flagellar detachment in C.reinhardtii cells.

Transgenic Chlamydomonas Expressing Human Transient Receptor Potential Ankyrin 1 (TRPA1) Channels to Assess the Effect of Agonists and Antagonists.

Transient receptor potential ankyrin 1 (TRPA1) channel is an ion channel whose gating is controlled by agonists, such as allyl isothiocyanate (AITC), and temperature. Since TRPA1 is associated with various disease symptoms and chemotherapeutic side effects, it is a frequent target of drug development. To facilitate the screening of TRPA1 agonists and antagonists, this study aimed to develop a simple bioassay for TRPA1 activity. To this end, transgenic Chlamydomonas reinhardtii expressing human TRPA1 was constructed. The transformants exhibited positive phototaxis at high temperatures (≥20°C) but negative phototaxis at low temperatures (≤15°C); wild-type cells showed positive phototaxis at all temperatures examined. In the transgenic cells, negative phototaxis was inhibited by TRPA1 antagonists, such as HC030031, A-967079, and AP18, at low temperatures. Negative phototaxis was induced by TRPA1 agonists, such as icilin and AITC, at high temperatures. The effects of these agonists were blocked by TRPA1 antagonists. In wild-type cells, none of these substances had any effects on phototaxis. These results indicate that the action of TRPA1 agonists and antagonists can be readily assessed using the behavior of C. reinhardtii expressing human TRPA1 as an assessment tool.

Temperature-sensitive mutants of MscL mechanosensitive channel.

MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified. The L86P mutation occurred in the second transmembrane helix, TM2. Substitution of residues neighbouring L86 with proline also led to a Ts mutation, but the substitution of L86 with other amino acids did not result in a Ts phenotype, indicating that the Ts phenotype was due to a structural change of TM2 helix by the introduction of a proline residue. The D127V mutation was localized in the electrostatic belt of the bundle of cytoplasmic helices, indicating that stability of the pentameric bundle of the cytoplasmic helix affects MscL structure. Together, this study described a novel class of MscL mutations that were correlated with the thermodynamic stability of the MscL structure.

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS.

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.

Thermotaxis in Chlamydomonas is brought about by membrane excitation and controlled by redox conditions.

Temperature is physiologically critical for all living organisms, which cope with temperature stress using metabolic and behavioral responses. In unicellular and some multicellular organisms, thermotaxis is a behavioral response to avoid stressful thermal environments and promote accumulation in an optimal thermal environment. In this study, we examined whether Chlamydomonas reinhardtii, a unicellular green alga, demonstrated thermotaxis. We found that between 10 °C and 30 °C, Chlamydomonas cells migrated toward lower temperatures independent of cultivation temperature. Interestingly, when we applied reagents to change intracellular reduction-oxidation (redox) conditions, we saw that thermotaxis was enhanced, suppressed, or reversed, depending on the redox conditions and cultivation temperature. Thermotaxis was almost absent in ppr2 and ppr3 mutants, which cannot swim backward because of a defect in generating calcium current in flagella. The frequency of spontaneous backward swimming was lower at more favorable temperature, suggesting a pivotal role of spontaneous backward swimming generated by flagellar membrane excitation.

Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain.

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from -40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at -60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas.

The motility of cilia and flagella of eukaryotic cells is controlled by second messengers such as Ca(2+), cAMP, and cGMP. In this study, the cAMP-dependent control of flagellar bending of Chlamydomonas is investigated by applying cAMP through photolysis of 4,5-dimethoxy-2-nitrobenzyl adenosine 3',5'-cyclicmonophosphate (caged cAMP). When cAMP is applied to demembranated and reactivated cells, cells begin to swim with a larger helical path. This change is due to a larger turn about the axis normal to the anterior-posterior axis, indicating an increased imbalance in the propulsive forces generated by the cis-flagellum (flagellum nearer to the eyespot) and trans-flagellum (flagellum farther from the eyespot). Consistently, when cAMP is applied to isolated axonemes, some axonemes show attenuated motility whereas others do not. Axonemes from uni1 mutants, which have only trans-flagella, do not respond to cAMP. These observations indicate that cAMP controls the balance of the forces generated by cis- and trans-flagella in Chlamydomonas.

A cytoplasmic helix is required for pentamer formation of the Escherichia coli MscL mechanosensitive channel.

Many membrane proteins such as ion channels are oligomers, but the determinants of the degree of oligomerization are not fully understood. Mechanosensitive channel with large conductance (MscL), which is ubiquitous in bacteria, is a homopentamer with two transmembrane helices and a cytoplasmic helix in each subunit. The carboxyl-terminal cytoplasmic helices assemble into a pentameric bundle that resembles cartilage oligomeric matrix protein. To address the role of cytoplasmic helices in the pentamer formation of Escherichia coli MscL, we generated MscL constructs with various deletions at the carboxyl terminus and translated them in a cell-free system. Deletions of Leu-129 and the downstream sequence resulted in formation of various oligomers without preference to pentamers, suggesting that nearly the whole cytoplasmic helix is required for MscL pentamer formation.

The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding.

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1-TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1-TM2 shaft and the state of the associated hollow cytoplasmic domain ("cage"). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the ß domain of the cage. We found that mutations destabilizing the TM3b-ß interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response.

A key molecule of sensing machineries essential for survival upon hypo-osmotic shock is the mechanosensitive channel. The bacterial mechanosensitive channel MscS functions directly for this purpose by releasing cytoplasmic solutes out of the cell, whereas plant MscS homologues are found to function in chloroplast organization. Here we show that the fission yeast MscS homologues, designated Msy1 and Msy2, participate in the hypo-osmotic shock response by a mechanism different from that operated by the bacterial MscS. Upon hypo-osmotic shock, msy2(-) and msy1(-) msy2(-) cells display greater cell swelling than wild-type cells and undergo cell death. Cell swelling precedes an intracellular Ca(2+) increase, which was greater in msy1(-) and msy1(-) msy2(-) cells than in wild-type cells. Fluorescent microscopy showed that Msy1 and Msy2 localize mainly to the endoplasmic reticulum. These observations suggest that organellar Msy1 and Msy2 regulate intracellular Ca(2+) and cell volume for survival upon hypo-osmotic shock.

Mechanoreception in motile flagella of Chlamydomonas.

Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted.

Mechanosensitivity of ion channels based on protein-lipid interactions.

Ion channels form a group of membrane proteins that pass ions through a pore beyond the energy barrier of the lipid bilayer. The structure of the transmembrane segment of membrane proteins is influenced by the charges and the hydrophobicity of the surrounding lipids and the pressure on its surface. A mechanosensitive channel is specifically designed to change its conformation in response to changes in the membrane pressure (tension). However, mechanosensitive channels are not the only group that is sensitive to the physical environment of the membrane: voltage-gated channels are also amenable to the lipid environment. In this article, we review the structure and gating mechanisms of the mechanosensitive channels and voltage-gated channels and discuss how their functions are affected by the physical properties of the lipid bilayer.