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Ching Kung

Publications and source records attributed to Ching Kung.

12 recordsLinked to original sources
PubMed2005

A possible unifying principle for mechanosensation.

Of Aristotle's five senses, we know that sight, smell and much of taste are initiated by ligands binding to G-protein-coupled receptors; however, the mechanical sensations of touch and hearing remain without a clear understanding of their molecular basis. Recently, the relevant force-transducing molecules--the mechanosensitive ion channels--have been identified. Such channel proteins purified from bacteria sense forces from the lipid bilayer in the absence of other proteins. Recent evidence has shown that lipids are also intimately involved in opening and closing the mechanosensitive channels of fungal, plant and animal species.

Animals↗
PubMed2005

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.

Archaea↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

Microbial mechanosensation.

Because bacterial mechanosensitive channels have been cloned, purified, crystallized and subjected to a genetic, biochemical and physical scrutiny, they have become the current structural models of mechanosensation to atomic detail. The key observation, supported by recent mutagenesis studies, is that these channels receive stretch force directly through the lipid bilayer at the interface levels bearing highest tension. Indeed, simulations of mechanosensitive channels steered by strategically applied bilayer stretch forces show channel opening. Our understanding of the gating energetics and trajectory are continually being refined by the combination of approaches applied. In addition, new microbial mechanosensitive channels from the TRP family have been characterized in yeasts. Unified by fundamental biophysical principles of gating, mechanosensitive channels provide broad insight into protein-membrane interactions and the role of hydrophobic hydration in gating.

Animals↗
PubMed2004

Channels in microbes: so many holes to fill.

Among players in neurobiology, ion channels are the demigods that underlie all our senses, behaviour and intelligence. In animals, these 'gated pores' detect ligands, voltage, heat or stretch forces and emit electric or ionic signals. Patch clamp and genome sequencing now show that nearly all microbes also have these 'smart' molecules. Microbial channel proteins have yielded crystal structures so dear to neuroscientists. However, their natural roles in microbial physiology remain largely unknown. The intellectual and technical schisms between 'neuro' and 'micro' biology must be bridged before we know how we became so smart, and whether microbes are just as smart.

Bacterial Physiological Phenomena↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2003

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.

Base Sequence↗
PubMed2003

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.

Alternative Splicing↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

Gating the bacterial mechanosensitive channel MscL invivo.

YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19-->Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET(+), thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an approximately 800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19-->Cys MscL in the presence of YggB, but a downshock of only approximately 400 mmol/kg is required in the absence of YggB. In parallel, Leu-19-->Cys MscL, stretched open by large sustained suction in the presence of MTSET(+) in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.

Bacterial Proteins↗
PubMed2002

Calmodulin as an ion channel subunit.

A surprising variety of ion channels found in a wide range of species from Homo to Paramecium use calmodulin (CaM) as their constitutive or dissociable Ca(2+)-sensing subunits. The list includes voltage-gated Ca(2+) channels, various Ca(2+)- or ligand-gated channels, Trp family channels, and even the Ca(2+)-induced Ca(2+) release channels from organelles. Our understanding of CaM chemistry and its relation to enzymes has been instructive in channel research, yet the intense study of CaM regulation of ion channels has also revealed unexpected CaM chemistry. The findings on CaM channel interactions have indicated the existence of secondary interaction sites in addition to the primary CaM-binding peptides and the functional differences between the N- and C-lobes of CaM. The study of CaM in channel biology will figure into our understanding on how this uniform, universal, vital, and ubiquitous Ca(2+) decoder coordinates the myriad local and global cell physiological transients.

Animals↗