In vitro and in vivo characterization of modulation of the vacuolar cation channel TRPY1 from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses a transient receptor potential (TRP) channel homolog TRPY1 in its vacuolar membrane, considered to be an ancestral TRP channel. So far, studies have focused on the channel properties of TRPY1, but its regulation and physiologic role remained to be elucidated. Here, we investigated TRPY1 channel function in vitro and in vivo. Patch-clamp recording of TRPY1 in yeast vacuolar membranes showed that Ca2+ on the lumen side inhibited TRPY1-mediated channel activity, whereas luminal Zn2+ increased the currents. TRPY1 was activated in the presence of a reducing agent, 2-mercaptoethanol. The cysteine at position 624 was identified as the target for this activating action. This activation was independent of the presence of cytosolic Ca2+ . The amplitude of TRPY1-mediated current was reduced by addition of phosphatidylinositol 3-phosphate on the cytosolic side but not by phosphatidylinositol (PI) or phosphatidylinositol 3,5-phosphate. Measurement of the transient Ca2+ increase in response to hyper-osmotic shock in several yeast mutants defective in different steps of the PI phosphate biogenesis pathway supported this interpretation. Addition of a microtubule inhibitor strongly decreased the transient cytosolic Ca2+ increase upon hyper-osmotic shock. Taken together, the data indicate that the vacuolar TRPY1 Ca2+ channel mediates the perception of cytosolic signals that were induced by external changes in osmolarity, and participates in the modulation of cytosolic calcium signaling through Ca2+ release from the vacuole to maintain intracellular Ca2+ homeostasis in yeast.

L-Glutamate secretion by the N-terminal domain of the Corynebacterium glutamicum NCgl1221 mechanosensitive channel.

The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1-286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287-533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221-232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420-533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel.

Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Glutamate is excreted across the cytoplasmic membrane through the NCgl1221 channel of Corynebacterium glutamicum by passive diffusion.

The NCgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (Glu) overproduction by Corynebacterium glutamicum, but direct evidence of Glu excretion through this channel has not yet been provided. In this study, by electrophysiological methods, we found direct evidence of Glu excretion through this channel by passive diffusion. We found that the introduction into Phe-producing Escherichia coli of mutant NCgl1221 genes that induce Glu overproduction by C. glutamicum improved productivity. This suggests a low-substrate preference of this channel, indicates its potential as a versatile exporter, and more broadly, indicates the potential of exporter engineering.

Patch clamp analysis of the respiratory chain in Bacillus subtilis.

Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

The protein encoded by NCgl1221 in Corynebacterium glutamicum functions as a mechanosensitive channel.

The function of the NCgl1221-encoded protein of Corynebacterium glutamicum was analyzed using Bacillus subtilis as host because a method for preparing the giant provacuole required for electrophysiological studies has been established. Expression of NCgl1221 in a strain deficient in mscL and ykuT, both of which encode mechanosensitive channels, resulted in an 8.9-fold higher cell survival rate upon osmotic downshock than the control. Electrophysiological investigation showed that the giant provacuole prepared from this strain, expressing NCgl1221, exhibited significantly higher pressure-dependent conductance than the control. These findings show that the NCgl1221-encoded protein functions as a mechanosensitive channel.

Electrophysiological properties of NtTPK1 expressed in yeast tonoplast.

The patch clamp technique using enlarged yeast tonoplasts is an effective approach to characterizing the Nicotiana tabacum plant tonoplast K+ channel, NtTPK1. We report here that the NtTPK1-mediated currents comprise two phase components, both which were found to be highly selective for K+ over Na+ and Cl-.

Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts.

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles.

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M). The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined. An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m). The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed.

A modified model for non-newtonian viscosity behavior of Aureobasidium pullulans culture fluid.

The culture fluid of the fungus Aureobasidium pullulans and the exopolysaccharide solution obtained by removal of the microbial cells exhibit a marked shear dependence of viscosity. The viscosity in a high shear rate region was a little higher than that predicted by a non-Newtonian viscosity equation derived previously on the basis of the concept of traveling force. In a sample exhibiting such high shear rate dependence, a hydrodynamic effect based on the fluid structure of the binding of contacting polymers and suspended microbial cells on viscosity becomes comparatively significant. A model for the shear rate dependence of the viscosity is needed to elucidate the mechanism of the viscosity behavior. A term concerning the increase in viscosity caused by the binding of polymers and the microbial cells suspended in a medium was added to the previous viscosity equation. The experimental shear dependence of the viscosity was well simulated by the modified viscosity equation.