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Article ; Online: A channelopathy mechanism revealed by direct calmodulin activation of TrpV4.

Loukin, Stephen H / Teng, Jinfeng / Kung, Ching

Proceedings of the National Academy of Sciences of the United States of America

2015 Volume 112, Issue 30, Page(s) 9400–9405

Abstract: Ca(2+)-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca(2+) increases TrpV4 activity through Ca(2+)-CaM binding to a C-terminal Ca(2+ ...

Abstract	Ca(2+)-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca(2+) increases TrpV4 activity through Ca(2+)-CaM binding to a C-terminal Ca(2+)-CaM binding domain (CBD). We confirmed this model by directly presenting Ca(2+)-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca(2+)-CaM. Deleting a 10-residue upstream peptide (Δ795-804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca(2+)-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca(2+)-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.
MeSH term(s)	Alleles ; Amino Acid Sequence ; Animals ; Binding Sites ; Bone Diseases/genetics ; Bone Diseases/physiopathology ; Calcium/chemistry ; Calmodulin/chemistry ; Channelopathies/physiopathology ; Chelating Agents/chemistry ; Gene Expression Profiling ; Humans ; Ion Channel Gating ; Molecular Sequence Data ; Mutation ; Oocytes/cytology ; Protein Binding/genetics ; Protein Structure, Tertiary ; RNA, Complementary/metabolism ; Sequence Homology, Amino Acid ; TRPV Cation Channels/genetics ; TRPV Cation Channels/physiology ; Xenopus laevis
Chemical Substances	Calmodulin ; Chelating Agents ; RNA, Complementary ; TRPV Cation Channels ; TRPV4 protein, human ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2015-07-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1510602112
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Article: A channelopathy mechanism revealed by direct calmodulin activation of TrpV4

Loukin, Stephen H / Jinfeng Teng / Ching Kung

Proceedings of the National Academy of Sciences of the United States of America. 2015 July 28, v. 112, no. 30

2015

Abstract: Over 50 mutations in the ion channel Transient Receptor Potential vanilloid subtype 4 (TrpV4) cause diseases ranging from dwarfism to prenatal death. We previously examined 14 mutant channels and found them to leak. Ca Â²âº encourages TrpV4 opening ... ...

Abstract	Over 50 mutations in the ion channel Transient Receptor Potential vanilloid subtype 4 (TrpV4) cause diseases ranging from dwarfism to prenatal death. We previously examined 14 mutant channels and found them to leak. Ca Â²âº encourages TrpV4 opening through calmodulin (CaM). Here, we examined two channels mutated in close proximity to the Ca Â²âº-CaMâbinding domain. They not only leak but also are greatly reduced in activation by Ca Â²âº-CaM compared with the wild-type or other mutant channels. These mutations likely define an autoinhibitory domain that keeps the channel closed, to which adjacent detachable Ca Â²âº-CaM binding interferes with this inhibition. The scattered disease alleles may all make the channel leak but apparently by different means, including the loss of an autoinhibition shown here. Ca Â²âº-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca Â²âº increases TrpV4 activity through Ca Â²âº-CaM binding to a C-terminal Ca Â²âº-CaM binding domain (CBD). We confirmed this model by directly presenting Ca Â²âº-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca Â²âº-CaM. Deleting a 10-residue upstream peptide (Î795â804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca Â²âº-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca Â²âº-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.
Keywords	alleles ; calcium ; calmodulin ; death ; mutants ; mutation ; transient receptor potential channels ; skeletal dysplasia ; ion channel ; autoinhibition ; TrpV1
Language	English
Dates of publication	2015-0728
Size	p. 9400-9405.
Publishing place	National Academy of Sciences
Document type	Article
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1510602112
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Article ; Online: A competing hydrophobic tug on L596 to the membrane core unlatches S4-S5 linker elbow from TRP helix and allows TRPV4 channel to open.

Teng, Jinfeng / Loukin, Stephen H / Anishkin, Andriy / Kung, Ching

Proceedings of the National Academy of Sciences of the United States of America

2016 Volume 113, Issue 42, Page(s) 11847–11852

Abstract: We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure- ... ...

Abstract	We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model-an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-of-function (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.
MeSH term(s)	Amino Acid Sequence ; Amino Acids/chemistry ; Animals ; Gain of Function Mutation ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Ion Channel Gating/drug effects ; Lipids/chemistry ; Loss of Function Mutation ; Membranes/chemistry ; Membranes/metabolism ; Models, Molecular ; Phenotype ; Protein Conformation ; Protein Interaction Domains and Motifs ; Structure-Activity Relationship ; TRPV Cation Channels/agonists ; TRPV Cation Channels/chemistry ; TRPV Cation Channels/genetics ; TRPV Cation Channels/metabolism ; Xenopus ; Yeasts/genetics ; Yeasts/metabolism
Chemical Substances	Amino Acids ; Lipids ; TRPV Cation Channels
Language	English
Publishing date	2016-10-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1613523113
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Article ; Online: The force-from-lipid (FFL) principle of mechanosensitivity, at large and in elements.

Teng, Jinfeng / Loukin, Stephen / Anishkin, Andriy / Kung, Ching

Pflugers Archiv : European journal of physiology

2014 Volume 467, Issue 1, Page(s) 27–37

Abstract: Focus on touch and hearing distracts attention from numerous subconscious force sensors, such as the vital control of blood pressure and systemic osmolarity, and sensors in nonanimals. Multifarious manifestations should not obscure invariant and ... ...

Abstract	Focus on touch and hearing distracts attention from numerous subconscious force sensors, such as the vital control of blood pressure and systemic osmolarity, and sensors in nonanimals. Multifarious manifestations should not obscure invariant and fundamental physicochemical principles. We advocate that force from lipid (FFL) is one such principle. It is based on the fact that the self-assembled bilayer necessitates inherent forces that are large and anisotropic, even at life's origin. Functional response of membrane proteins is governed by bilayer force changes. Added stress can redirect these forces, leading to geometric changes of embedded proteins such as ion channels. The FFL principle was first demonstrated when purified bacterial mechanosensitive channel of large conductance (MscL) remained mechanosensitive (MS) after reconstituting into bilayers. This key experiment has recently been unequivocally replicated with two vertebrate MS K2p channels. Even the canonical Kv and the Drosophila canonical transient receptor potentials (TRPCs) have now been shown to be MS in biophysical and in physiological contexts, supporting the universality of the FFL paradigm. We also review the deterministic role of mechanical force during stem cell differentiation as well as the cell-cell and cell-matrix tethers that provide force communications. In both the ear hair cell and the worm's touch neuron, deleting the cadherin or microtubule tethers reduces but does not eliminate MS channel activities. We found no evidence to distinguish whether these tethers directly pull on the channel protein or a surrounding lipid platform. Regardless of the implementation, pulling tether tenses up the bilayer. Membrane tenting is directly visible at the apexes of the stereocilia.
MeSH term(s)	Cell Membrane/physiology ; Ion Channel Gating/physiology ; Ion Channels/physiology ; Lipid Bilayers/metabolism ; Mechanotransduction, Cellular/physiology ; Membrane Fluidity/physiology ; Models, Biological ; Stress, Mechanical
Chemical Substances	Ion Channels ; Lipid Bilayers
Language	English
Publishing date	2014-06-03
Publishing country	Germany
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	6380-0
ISSN	1432-2013 ; 0031-6768
ISSN (online)	1432-2013
ISSN	0031-6768
DOI	10.1007/s00424-014-1530-2
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Article ; Online: L596-W733 bond between the start of the S4-S5 linker and the TRP box stabilizes the closed state of TRPV4 channel.

Teng, Jinfeng / Loukin, Stephen H / Anishkin, Andriy / Kung, Ching

Proceedings of the National Academy of Sciences of the United States of America

2015 Volume 112, Issue 11, Page(s) 3386–3391

Abstract: Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid ... ...

Abstract	Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4-S5 linker helix. By homology modeling, we identified the corresponding L596-W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4-S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar-nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.
MeSH term(s)	Amino Acid Substitution ; Animals ; Humans ; Ion Channel Gating ; Leucine/chemistry ; Mutant Proteins/chemistry ; Mutant Proteins/metabolism ; Mutation/genetics ; Oocytes ; Phenotype ; Protein Stability ; Protein Structure, Secondary ; Saccharomyces cerevisiae/growth & development ; Structure-Activity Relationship ; TRPV Cation Channels/chemistry ; TRPV Cation Channels/genetics ; TRPV Cation Channels/metabolism ; Tryptophan/chemistry ; Xenopus
Chemical Substances	Mutant Proteins ; TRPV Cation Channels ; TRPV4 protein, human ; Tryptophan (8DUH1N11BX) ; Leucine (GMW67QNF9C)
Language	English
Publishing date	2015-03-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1502366112
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Article ; Online: Yeast luminometric and Xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of TRPV4.

Teng, Jinfeng / Loukin, Stephen / Zhou, Xinliang / Kung, Ching

Journal of visualized experiments : JoVE

2013 , Issue 82

Abstract: TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies ... ...

Abstract	TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca(2+), released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10(-6) to 10(-4) A) currents from oocytes are much larger than the subnano- to nanoAmpere (10(-10) to 10(-9) A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.
MeSH term(s)	Animals ; Biomechanical Phenomena ; Luminescent Measurements/methods ; Oocytes/metabolism ; Oocytes/physiology ; Patch-Clamp Techniques/methods ; Plasmids/genetics ; Polymerase Chain Reaction/methods ; Rats ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; TRPC Cation Channels/biosynthesis ; TRPC Cation Channels/genetics ; Transgenes ; Xenopus laevis/metabolism ; Xenopus laevis/physiology
Chemical Substances	TRPC Cation Channels ; TRPC4 ion channel
Language	English
Publishing date	2013-12-31
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/50816
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Article ; Online: Feeling the hidden mechanical forces in lipid bilayer is an original sense.

Anishkin, Andriy / Loukin, Stephen H / Teng, Jinfeng / Kung, Ching

Proceedings of the National Academy of Sciences of the United States of America

2014 Volume 111, Issue 22, Page(s) 7898–7905

Abstract: Life's origin entails enclosing a compartment to hoard material, energy, and information. The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous domains. The self-assembled lipid bilayer comes with a ... ...

Abstract	Life's origin entails enclosing a compartment to hoard material, energy, and information. The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous domains. The self-assembled lipid bilayer comes with a set of properties including its strong anisotropic internal forces that are chemically or physically malleable. Added bilayer stretch can alter force vectors on embedded proteins to effect conformational change. The force-from-lipid principle was demonstrated 25 y ago when stretches opened purified Escherichia coli MscL channels reconstituted into artificial bilayers. This reductionistic exercise has rigorously been recapitulated recently with two vertebrate mechanosensitive K(+) channels (TREK1 and TRAAK). Membrane stretches have also been known to activate various voltage-, ligand-, or Ca(2+)-gated channels. Careful analyses showed that Kv, the canonical voltage-gated channel, is in fact exquisitely sensitive even to very small tension. In an unexpected context, the canonical transient-receptor-potential channels in the Drosophila eye, long presumed to open by ligand binding, is apparently opened by membrane force due to PIP2 hydrolysis-induced changes in bilayer strain. Being the intimate medium, lipids govern membrane proteins by physics as well as chemistry. This principle should not be a surprise because it parallels water's paramount role in the structure and function of soluble proteins. Today, overt or covert mechanical forces govern cell biological processes and produce sensations. At the genesis, a bilayer's response to osmotic force is likely among the first senses to deal with the capricious primordial sea.
MeSH term(s)	Animals ; Biological Evolution ; Humans ; Ion Channel Gating/physiology ; Lipid Bilayers/chemistry ; Mechanotransduction, Cellular/physiology ; Origin of Life ; Osmotic Pressure ; Stress, Mechanical ; Touch/physiology
Chemical Substances	Lipid Bilayers
Language	English
Publishing date	2014-05-21
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1313364111
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Article ; Online: Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations.

Loukin, Stephen / Su, Zhenwei / Kung, Ching

PloS one

2011 Volume 6, Issue 5, Page(s) e19533

Abstract: TRPV4 is a mechanically activated Ca(2+)-passing channel implicated in the sensing of forces, including those acting on bones. To date, 33 mutations are known to affect human bone development to different extents. The spectrum of these skeletal ... ...

Abstract	TRPV4 is a mechanically activated Ca(2+)-passing channel implicated in the sensing of forces, including those acting on bones. To date, 33 mutations are known to affect human bone development to different extents. The spectrum of these skeletal dysplasias (SD) ranges from dominantly inherited mild brachylomia (BO) to neonatal lethal forms of metatropic dysplasia (MD). Complexities of the results from fluorescence and electrophysiological studies have led to questions on whether channel activity is a good predictor of disease severity. Here we report on a systematic examination of 14 TRPV4 mutant alleles covering the entire SD spectrum. Expressed in Xenopus oocyte and without any stimulation, the wild-type channel had a ~1% open probability (Po) while those of most of the lethal MD channels approached 100%. All mutant channels had higher basal open probabilities, which limited their further increase by agonist or hypotonicity. The magnitude of this limitation revealed a clear correlation between the degree of over-activity (the molecular phenotype) and the severity of the disease over the entire spectrum (the biological phenotype). Thus, while other factors are at play, our results are consistent with the increased TRPV4 basal activity being a critical determinant of the severity of skeletal dysplasia. We discuss how the channel over-activity may lead to the "gain-of-function" phenotype and speculate that the function of wild-type TRPV4 may be secondary in normal bone development but crucial in an acute process such as fracture repair in the adult.
MeSH term(s)	Animals ; Humans ; Oocytes ; Osteochondrodysplasias/etiology ; Osteochondrodysplasias/genetics ; Osteochondrodysplasias/metabolism ; Osteochondrodysplasias/pathology ; Patch-Clamp Techniques ; TRPV Cation Channels/genetics ; TRPV Cation Channels/metabolism ; Xenopus laevis
Chemical Substances	TRPV Cation Channels ; TRPV4 protein, human
Language	English
Publishing date	2011-05-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0019533
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Article ; Online: Forward genetic analysis reveals multiple gating mechanisms of TRPV4.

Loukin, Stephen / Su, Zhenwei / Zhou, Xinliang / Kung, Ching

The Journal of biological chemistry

2010 Volume 285, Issue 26, Page(s) 19884–19890

Abstract: TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a ... ...

Abstract	TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4alphaPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca(2+) block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.
MeSH term(s)	Alleles ; Animals ; Calcium/pharmacology ; Female ; Galactose/pharmacology ; Glucose/pharmacology ; Hypotonic Solutions/pharmacology ; Ion Channel Gating/genetics ; Ion Channel Gating/physiology ; Membrane Potentials/drug effects ; Mutation ; Oocytes/physiology ; Patch-Clamp Techniques ; Phorbol Esters/pharmacology ; Rats ; Saccharomyces cerevisiae/drug effects ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/growth & development ; TRPV Cation Channels/genetics ; TRPV Cation Channels/physiology ; Xenopus
Chemical Substances	Hypotonic Solutions ; Phorbol Esters ; TRPV Cation Channels ; Trpv4 protein, rat ; phorbol-12,13-didecanoate (24928-17-4) ; Glucose (IY9XDZ35W2) ; Calcium (SY7Q814VUP) ; Galactose (X2RN3Q8DNE)
Language	English
Publishing date	2010-04-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M110.113936
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Article: Forward Genetic Analysis Reveals Multiple Gating Mechanisms of TRPV4

Loukin, Stephen / Su, Zhenwei / Zhou, Xinliang / Kung, Ching

Journal of biological chemistry. 2010 June 25, v. 285, no. 26

2010

Abstract	TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4αPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca²⁺ block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.
Language	English
Dates of publication	2010-0625
Size	p. 19884-19890.
Publishing place	American Society for Biochemistry and Molecular Biology
Document type	Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
Database	NAL-Catalogue (AGRICOLA)

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