Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.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.
???displayArticle.pubmedLink???
24637628 ???displayArticle.pmcLink???PMC4396860 ???displayArticle.link???J Vis Exp ???displayArticle.grants???[+]
Batiza,
Yeast respond to hypotonic shock with a calcium pulse.
1996, Pubmed
Batiza,
Yeast respond to hypotonic shock with a calcium pulse.
1996,
Pubmed Brown,
Patch clamp recording of ion channels expressed in Xenopus oocytes.
2008,
Pubmed
,
Xenbase Camacho,
Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia.
2010,
Pubmed Delany,
Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.
2001,
Pubmed Denis,
Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.
2002,
Pubmed Everaerts,
The vanilloid transient receptor potential channel TRPV4: from structure to disease.
2010,
Pubmed Gao,
Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.
2003,
Pubmed Goldin,
Preparation of RNA for injection into Xenopus oocytes.
1992,
Pubmed
,
Xenbase Hamill,
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
1981,
Pubmed Kang,
Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation.
2012,
Pubmed Krakow,
Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia.
2009,
Pubmed Lamandé,
Mutations in TRPV4 cause an inherited arthropathy of hands and feet.
2011,
Pubmed Liedtke,
Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.
2000,
Pubmed Loukin,
Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations.
2011,
Pubmed
,
Xenbase Loukin,
Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.
1997,
Pubmed
,
Xenbase Loukin,
A genome-wide survey suggests an osmoprotective role for vacuolar Ca2+ release in cell wall-compromised yeast.
2008,
Pubmed Loukin,
Lipid perturbations sensitize osmotic down-shock activated Ca2+ influx, a yeast "deletome" analysis.
2007,
Pubmed Loukin,
Forward genetic analysis reveals multiple gating mechanisms of TRPV4.
2010,
Pubmed
,
Xenbase Loukin,
Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids.
2009,
Pubmed Loukin,
Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force.
2010,
Pubmed
,
Xenbase Maroto,
TRPC1 forms the stretch-activated cation channel in vertebrate cells.
2005,
Pubmed
,
Xenbase Nilius,
The puzzle of TRPV4 channelopathies.
2013,
Pubmed Nilius,
The transient receptor potential family of ion channels.
2011,
Pubmed Ramsey,
An introduction to TRP channels.
2006,
Pubmed Rock,
Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia.
2008,
Pubmed Strotmann,
OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity.
2000,
Pubmed Thorneloe,
An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.
2012,
Pubmed Vincent,
Identification and characterization of novel TRPV4 modulators.
2009,
Pubmed
,
Xenbase Voets,
Molecular determinants of permeation through the cation channel TRPV4.
2002,
Pubmed Vriens,
Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.
2004,
Pubmed Watanabe,
Modulation of TRPV4 gating by intra- and extracellular Ca2+.
2003,
Pubmed Watanabe,
Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels.
2003,
Pubmed Willette,
Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.
2008,
Pubmed Wissenbach,
Trp12, a novel Trp related protein from kidney.
2000,
Pubmed
