The core domain as the force sensor of the yeast mechanosensitive TRP channel.

Stretch-activated conductances are commonly encountered in careful electric recordings. Those of known proteins (TRP, MscL, MscS, K(2p), Kv, etc.) all share a core, which houses the ion pathway and the gate, but no recognizable force-sensing domain. Like animal TRPs, the yeast TRPY1 is polymodal, activated by stretch force, Ca(2+), etc. To test whether its S5-S6 core senses the stretch force, we tried to uncouple it from the peripheral domains by strategic peptide insertions to block the covalent core-periphery interactions. Insertion of long unstructured peptides should distort, if not disrupt, protein structures that transmit force. Such insertions between S6 and the C-terminal tail largely removed Ca(2+) activation, showing their effectiveness. However, such insertions as well as those between S5 and the N-terminal region, which includes S1-S4, did not significantly alter mechanosensitivity. Even insertions at both locations flanking the S5-S6 core did not much alter mechanosensitivity. Tryptophan scanning mutations in S5 were also constructed to perturb possible noncovalent core-periphery contacts. The testable tryptophan mutations also have little or no effects on mechanosensitivity. Boltzmann fits of the wild-type force-response curves agree with a structural homology model for a stretch-induced core expansion of ~2 nm(2) upon opening. We hypothesize that membrane tension pulls on S5-S6, expanding the core and opening the TRPY1 gate. The core being the major force sensor offers the simplest, though not the only, explanation of why so many channels of disparate designs are mechanically sensitive. Compared with the bacterial MscL, TRPY1 is much less sensitive to force, befitting a polymodal channel that relies on multiple stimuli.

Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force.

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 force-related 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.

The use of yeast to understand TRP-channel mechanosensitivity.

Mechanosensitive (MS) ion channels likely underlie myriad force-sensing processes, from basic osmotic regulation to specified sensations of animal hearing and touch. Albeit important, the molecular identities of many eukaryotic MS channels remain elusive, let alone their working mechanisms. This is in stark contrast to our advanced knowledge on voltage- or ligand-sensitive channels. Several members of transient receptor potential (TRP) ion channel family have been implicated to function in mechanosensation and are recognized as promising candidate MS channels. The yeast TRP homolog, TRPY1, is clearly a first-line force transducer. It can be activated by hypertonic shock in vivo and by membrane stretch force in excised patches under patch clamp, making it a useful model for understanding TRP channel mechanosensitivity in general. TRPY1 offers two additional research advantages: (1) It has a large ( approximately 300 pS) unitary conductance and therefore a favorable S/N ratio. (2) Budding yeast allows convenient and efficient genetic and molecular manipulations. In this review, we focus on the current research of TRPY1 and discuss its prospect. We also describe the use of yeast as a system to express and characterize animal TRP channels.

Mechanical force and cytoplasmic Ca(2+) activate yeast TRPY1 in parallel.

The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca(2+). Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca(2+). Tens of micromolar Ca(2+) greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca(2+)-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca(2+) activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca(2+) activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure-function model in which Ca(2+) binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.

Ion channels in microbes.

Studies of ion channels have for long been dominated by the animalcentric, if not anthropocentric, view of physiology. The structures and activities of ion channels had, however, evolved long before the appearance of complex multicellular organisms on earth. The diversity of ion channels existing in cellular membranes of prokaryotes is a good example. Although at first it may appear as a paradox that most of what we know about the structure of eukaryotic ion channels is based on the structure of bacterial channels, this should not be surprising given the evolutionary relatedness of all living organisms and suitability of microbial cells for structural studies of biological macromolecules in a laboratory environment. Genome sequences of the human as well as various microbial, plant, and animal organisms unambiguously established the evolutionary links, whereas crystallographic studies of the structures of major types of ion channels published over the last decade clearly demonstrated the advantage of using microbes as experimental organisms. The purpose of this review is not only to provide an account of acquired knowledge on microbial ion channels but also to show that the study of microbes and their ion channels may also hold a key to solving unresolved molecular mysteries in the future.

Indole and other aromatic compounds activate the yeast TRPY1 channel.

The yeast TRPY1 (Yvc1p) channel is activated by membrane stretch to release vacuolar Ca2+ into the cytoplasm upon osmotic upshock. Exogenously added indole greatly enhances the upshock-induced Ca2+ release in vivo. Indole also reversibly activates the channels under patch clamp. A minimum of 10(-6)M Ca2+ is needed for membrane stretch force to open TPRY1, but indole activation appears to be Ca2+ independent. A deletion of 30 residues at the predicted cytoplasmic domain, 570-600Delta, renders TRPY1 insensitive to stretch force upto 10(-3)M Ca2+. Nonetheless, indole readily activates this mutant channel. Several other aromatic compounds, e.g. the antimicrobial parabens, also activate TRPY1. These compounds likely alter the innate forces in the lipid bilayer received by the channel.

A genome-wide survey suggests an osmoprotective role for vacuolar Ca2+ release in cell wall-compromised yeast.

In yeast, osmotic upshock causes a release of vacuolar Ca(2+) through the mechanosensitive transient receptor potential channel, Yvc1. We screened the collection of 4810 yeast gene deletants twice for alterations in this response in an attempt to find elements that regulate the amount of vacuolar Ca(2+) or the Yvc1 channel. Severe overresponders and underresponders to upshock were further scrutinized for their calcium content with (45)Ca and their Yvc1 electrophysiological activities under patch-clamp. The severe underresponders have lower calcium content but no change in Yvc1 activity. The strong overresponders, most of which are deleted of genes involved in cell wall metabolism, have higher calcium content. Wall mutations are known to up-regulate Ca(2+)-calcineurin-dependent genes. It appears that stress on the cell wall induces Ca(2+) accumulation, adaptively anticipating the need in defense or repair against future stress, including osmotic stress.

Yeast gain-of-function mutations reveal structure-function relationships conserved among different subfamilies of transient receptor potential channels.

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

Yeast screens show aromatic residues at the end of the sixth helix anchor transient receptor potential channel gate.

Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance, the arrangement and roles of the amino acids that constitute the TRP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca(2+). After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca(2+), indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one alpha-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed.

Patch clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia coli as a host.

Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K+ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K+, indicating MmaK forms a functional K+ conduit in the E. coli membrane in vivo. After enlarging these cells into giant spheroplasts, macro- and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and approximately 10-fold higher concentration of cGMP and manifests as an inwardly rectified, K+-specific current with a 10.8 pS unitary conductance at -100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics.

Lipid perturbations sensitize osmotic down-shock activated Ca2+ influx, a yeast "deletome" analysis.

Osmotic down shock causes an immediate influx of Ca2+ in yeast, likely through a membrane stretch-sensitive channel. To see how this channel is constituted and regulated, we screened the collection of 4,906 yeast gene deletants for major changes in this response by luminomtery. We discovered deletants that responded very strongly to much milder down shocks than wild-type required, but show little changes in up-shock response. Of all the possibilities (general metabolism, ion distribution, cytoskeleton, cell wall, membrane receptors, etc.), most of the over-responders turned out to be deleted of proteins functioning in the biogenesis of phospholipids, sphingolipids, or ergosterol. Other over-responders are annotated to have vesicular transport defects, traceable to lipid defects in some cases. The deletant lacking the de novo synthesis of phosphatidylcholine, opi3delta, is by far the strongest over-responder. opi3 deletion does not cause non-specific leakage but greatly sensitizes the force-sensing Ca2+-influx mechanism. Choline supplementation normalizes the opi3delta response. Thus, the osmotic-pressure induced stretch force apparently controls channel activities through lipids. This unbiased examination of the yeast genome supports the view that forces intrinsic to the bilayer are determined by the geometry of the lipids and these forces, in turn, govern the activities of proteins embedded therein.

Prokaryotic K(+) channels: from crystal structures to diversity.

The deep roots and wide branches of the K(+)-channel family are evident from genome surveys and laboratory experimentation. K(+)-channel genes are widespread and found in nearly all the free-living bacteria, archaea and eukarya. The conservation of basic structures and mechanisms such as the K(+) filter, the gate, and some of the gate's regulatory domains have allowed general insights on animal K(+) channels to be gained from crystal structures of prokaryotic channels. Since microbes are the great majority of life's diversity, it is not surprising that microbial genomes reveal structural motifs beyond those found in animals. There are open-reading frames that encode K(+)-channel subunits with unconventional filter sequences, or regulatory domains of different sizes and numbers not previously known. Parasitic or symbiotic bacteria tend not to have K(+) channels, while those showing lifestyle versatility often have more than one K(+)-channel gene. It is speculated that prokaryotic K(+) channels function to allow adaptation to environmental and metabolic changes, although the actual roles of these channels in prokaryotes are not yet known. Unlike enzymes in basic metabolism, K(+) channel, though evolved early, appear to play more diverse roles than revealed by animal research. Finding and sorting out these roles will be the goal and challenge of the near future.

Heterologously expressed fungal transient receptor potential channels retain mechanosensitivity in vitro and osmotic response in vivo.

The budding yeast Saccharomyces cerevisiae has a mechanosensitive channel, TrpY1, a member of the Trp superfamily of channels associated with various sensations. Upon a hyperosmotic shift, a yeast cell releases Ca(2+) from the vacuole to the cytoplasm through this channel. The TRPY1 gene has orthologs in other fungal genomes, including TRPY2 of Kluyveromyces lactis and TRPY3 of Candida albicans. We subcloned TRPY2 and TRPY3 and expressed them in the vacuole of S. cerevisiae deleted of TRPY1. The osmotically induced Ca(2+) transient was restored in vivo as reported by transgenic aequorin. Patch-clamp examination showed that the TrpY2 or the TrpY3 channel was similar to TrpY1 in unitary conductance, rectification properties, Ca(2+) sensitivity, and mechanosensitivity. The retention of mechanosensitivity of transient receptor potential channels in a foreign setting, shown here both in vitro and in vivo, implies that these mechanosensitive channels, like voltage-gated or ligand-gated channels, do not discriminate their settings. We discuss various mechanisms, including the possibility that stress from the lipid bilayer by osmotic force transmits forces to the transmembrane domains of these channels.

PAK paradox: Paramecium appears to have more K(+)-channel genes than humans.

K(+)-selective ion channels (K(+) channels) have been found in bacteria, archaea, eucarya, and viruses. In Paramecium and other ciliates, K(+) currents play an essential role in cilia-based motility. We have retrieved and sequenced seven closely related Paramecium K(+)-channel gene (PAK) sequences by using previously reported fragments. An additional eight unique K(+)-channel sequences were retrieved from an indexed library recently used in a pilot genome sequencing project. Alignments of these protein translations indicate that while these 15 genes have diverged at different times, they all maintain many characteristics associated with just one subclass of metazoan K(+) channels (CNG/ERG type). Our results indicate that most of the genes are expressed, because all predicted frameshifts and several gaps in the homolog alignments contain Paramecium intron sequences deleted from reverse transcription-PCR products. Some of the variations in the 15 genomic nucleotide sequences involve an absence of introns, even between very closely related sequences, suggesting a potential occurrence of reverse transcription in the past. Extrapolation from the available genome sequence indicates that Paramecium harbors as many as several hundred of this one type of K(+)-channel gene. This quantity is far more numerous than those of K(+)-channel genes of all types known in any metazoan (e.g., approximately 80 in humans, approximately 30 in flies, and approximately 15 in Arabidopsis). In an effort to understand this plurality, we discuss several possible reasons for their maintenance, including variations in expression levels in response to changes in the freshwater environment, like that seen with other major plasma membrane proteins in Paramecium.

Gain-of-function mutations indicate that Escherichia coli Kch forms a functional K+ conduit in vivo.

Although Kch of Escherichia coli is thought to be a K(+) channel by sequence homology, there is little evidence that it actually conducts K(+) ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria specifically sensitive to K(+) ions. Millimolar added K(+), but not Na(+) or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K(+)-filter sequence rescue the GOF mutant. The apparent K(+)-specific conduction through the 'loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K(+) concentration. Possible implications on the normal function of Kch are discussed.

The transient receptor potential channel on the yeast vacuole is mechanosensitive.

Ca2+ 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 Ca2+ 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 Ca2+ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca2+ concentration and is apparently independent of its known Ca2+ activation, which we now propose to be an amplification mechanism (Ca2+-induced Ca2+ 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.

An exchanger-like protein underlies the large Mg2+ current in Paramecium.

There are very few molecules known to transport Mg(2+) in eukaryotes. The membrane of Paramecium tetraurelia passes a large Mg(2+)-selective current and exhibits a corresponding backward swimming behavior. Both are missing in a group of mutants called eccentric. By sorting an indexed WT genomic library through microinjection into the macronucleus, we have isolated a DNA fragment that complements the eccentric mutations. The Mg(2+) currents and behavior are restored fully in the transformed cells. Surprisingly, the conceptually translated protein is not homologous to any known ion channel but instead has some similarity to K(+)-dependent Na(+)Ca(2+) exchangers. Exchangers are either electrically silent or only pass very small and slow currents compared with ion-channel currents. In light of recent ion-channel crystal structures and considering the need to have narrow ion-selective filters, we speculate on how an exchanger might evolve to show channel-like activities in special circumstances. The significance of finding the molecular basis of a Mg(2+)-specific pathway is also discussed.

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K+ channel.

Non-targeted mutagenesis studies of the yeast K(+) channel, TOK1, have led to identification of functional domains common to other cation channels as well as those so far not found in other channels. Among the latter is the ability of the carboxyl tail to prevent channel closure. Here, we show that the tail can fulfill this function in trans. Coexpression of the carboxyl tail with the tail-deleted channel core restores normal channel behavior A Ser/Thr-rich region at its amino end and an acidic stretch at its carboxyl end delineate the minimal region required for tail function. This region of 160 aa apparently forms a discrete functional domain. Interaction of this domain with the channel core is strong, being recalcitrant to removal from excised membrane patches by both high salt and reducing agents. Although the use of a cytoplasmic domain to regulate channel is common among animal channels, by using it as a "foot-in-the-door" to maintain open state appears unique to TOK1, the first fungal K(+) channel studied in depth.