We would like to thank Andrea Kremsreiter for excellent technical assistance. Work in our laboratory is supported by NIH GM096088 and the Vilas Trust of the University of Wisconsin - Madison.

1. Yeast Luminometry Methods
Use strain BYYT of Saccharomyces cerevisiae. It is a yvc1- tok1- derivative of BY4741 (MATa, ura3D0, his3D1, leu2D0, lys2D0, yvc1::HIS3, tok1::kanMX4). Transform cells with the leu-selectable aequorin-expressing plasmid pEVP1/Aeq and the ura-selectable rat-TRPV4-expressing plasmid p416GPDV4, as described in Batiza et al.26 and Loukin et al.24 Use standard methods of plasmid construct...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+human+vanilloid+receptor-like+protein,+VRL-2.">Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.</a> Physiol. Genomics. 4, 165-174 (2001).</li><li>Rock, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+mutations+in+TRPV4+cause+autosomal+dominant+brachyolmia.">Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia.</a> Nat. 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U.S.A. 101, 396-401 (2004).</li><li>Vincent, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+TRPV4+modulators.">Identification and characterization of novel TRPV4 modulators.</a> Biochem. Biophys. Res. Commun. 389, 490-494 (2009).</li><li>Willette, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+activation+of+the+transient+receptor+potential+vanilloid+subtype+4+channel+causes+endothelial+failure+and+circulatory+collapse:+Part+2.">Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.</a> J. Pharmacol. Exp. Ther. 326, 443-452 (2008).</li><li>Thorneloe, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orally+active+TRPV4+channel+blocker+prevents+and+resolves+pulmonary+edema+induced+by+heart+failure.">An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.</a> Sci. Transl. Med. 4, (2012).</li><li>Kang, S. S., Shin, S. H., Auh, C. K., Chun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+dysplasia+caused+by+a+constitutive+activated+transient+receptor+potential+vanilloid+4+(TRPV4)+cation+channel+mutation.">Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation.</a> Exp. Mol. Med. 44, 707-722 (2012).</li><li>Lamande, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+TRPV4+cause+an+inherited+arthropathy+of+hands+and+feet.">Mutations in TRPV4 cause an inherited arthropathy of hands and feet.</a> Nat. Genet. 43, 1142-1146 (2011).</li><li>Loukin, S., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Basal+Activity+Is+a+Key+Determinant+in+the+Severity+of+Human+Skeletal+Dysplasia+Caused+by+TRPV4+Mutations.">Increased Basal Activity Is a Key Determinant in the Severity of Human Skeletal Dysplasia Caused by TRPV4 Mutations.</a> PLoS One. 6, (2011).</li><li>Loukin, S. H., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotonic+shocks+activate+rat+TRPV4+in+yeast+in+the+absence+of+polyunsaturated+fatty+acids.">Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids.</a> FEBS Lett. 583, 754-758 (2009).</li><li>Loukin, S., Su, Z., Zhou, X., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+genetic+analysis+reveals+multiple+gating+mechanisms+of+TRPV4.">Forward genetic analysis reveals multiple gating mechanisms of TRPV4.</a> J. Biol. Chem. 285, 19884-19890 (2010).</li><li>Batiza, A. F., Schulz, T., Masson, P. 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The authors declare that they have no competing financial interests.

As stated in the Introduction, the methods commonly used to study TRPV4 functions sometimes resulted in inconsistencies and controversies. The two sets of methods described here offer some advantages and can complement the existing methods. While we describe only the studies on TRPV4, the methods may be extended to study other ion channels as well.
1. Yeast Luminometry Methods
Budding yeast has a native Ca2+-influx response to hypo-osmotic shock that is sen...

TRPs (transient receptor potentials) comprise seven subfamilies of cation channels that serve sensory functions1,2. In mammals, TRPV, the vanilloid subfamily of TRPs, has six varieties. TRPV4 (type 4)3,4 responds to heat, certain chemicals, osmotic swelling, or shear stress. The TRPV4 gene was repeatedly isolated by candidate-gene and/or expression cloning5-8. The latter method followed the gene product&#39;s response to hypo-osmolarity. TRPV4 is expressed in nearly all organs and functions in the development, physiology, or pathology of many disparate cell types3,4.
Striking are the &#62;50 human autosomal dominant TRPV4 mutations, causing peripheral neuropathies and/or skeletal dysplasias (abnormalities in skeletal development)9-11. The skeletal dysplasias range from mild brachyomia type 3 (type-3 dwarfism), spondylometaphyseal dysplasia Kozlowski type, to severe dysplasias, some causing neonatal or embryonic death. Though all manners of mechanisms seem possible, none explains the diversity, complexity, variability, and occasional overlaps of these diseases4.
Like other TRP channels1, TRPV4 is a tetramer. In rat or human TRPV4, each subunit is consisted of 871 residues. Its central element is the six transmembrane a helices (S1-S6), which are likely arranged in a manner similar to voltage-gated K+ channels. There, S1 to S4 form a peripheral domain and the S5 and S6 form the permeation pore domain. Between S5 and S6 of TRPV4 is a short pore helix followed by the sequence LDLFKLTIGMGDL, four of which presumably converge to form the cation filter. The 470-residue N-terminal cytoplasmic segment contains 6 ankyrin repeats, known to bind proteins or small ligands. The C-terminal 152-residue cytoplasmic segment includes a calmodulin-binding sequence among other possible sites that bind other elements3.
TRPV4 is a cation channel that essentially excludes anions1. While its physiological function is to transduce stimuli to Ca2+ influx, it is also permeable to other cations with an Eisenman IV permeability sequence, favoring divalent at a PCa : PNa ~7 12. Single-channel conductance rectifies at ~90 pS outward and ~40 pS inward6,13,14. Heterologously expressed current (below) can be activated by hypo-osmotic swelling, shear stress, or warmth15. It is also activated by polyunsaturated fatty acids16,17 and the synthetic phorbal ester 4&#945;PDD 18. At present, the most potent agonist is GSK1016790A 19 and antagonist is GSK2193874, effective in 10-9 to 10-8 M 20, both discovered by high-throughput, small-molecular screen.
Two key areas of TRPV4 research remain confusing: (1) Even as TRPV4 is largely studied for its mechanosensitivity, its molecular basis is controversial. One model describes hypo-osmolarity somehow activates phospholipase A2 (PLA2) to produce the polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which is converted to epoxyeicosatrienoic acid (EET) by an epoxygenase, and the binding of EET activates TRPV4 16,17. Yet, TRPV4 itself has been shown to directly respond to membrane stretch14 (below), providing a simpler explanation. (2) The TRPV4 mutant pathologies are bewildering. At the foundation, one needs to know whether the diseases are due to the loss, the reduction, or the increase of channel activities. Even here, the literature is far from clear. While multiple skeletal-dysplasia alleles were reported to have higher activities, (gain-of-function, GOF)4,21, several were reported to have reduced activities (loss-of-function, LOF)10,22. A systematic study of 14 alleles found them to all be GOF mutations (below)23. The claim that some are LOFs seems to contradict the phenotype of trpV4-/- knockout mouse, which are viable or fertile, with only minor defects, despite a complete loss of TRPV4 function.
A part of these controversies has methodological origin. Laboratories use different methods, or variants of one method, and employ different judging standards. Most commonly, TRPV4 is transiently expressed in cultured mammalian cells (HEK, CHO, HeLa) and the rise of internal [Ca2+] upon agonist or hypotonic stimulation is registered with the Ca2+-sensitive fluorescent dye, Fura-2. The over-reliance on this fluorometric assay has been criticized1. The expression level in different populations, the distribution therein, as well as the effective dye concentration, need to be controlled and documented. More reliable is the direct electrophysiological examinations. Even this, as commonly practiced, is also not without problems. Because the expression levels in individual cultured cells are difficult to control, whole-cell currents have large variations. Further, because the currents are small, reliable statistics will have to rely on large sample sizes, often not practical. Patch-clamp examinations have rarely been performed. Some such recordings show clusters of activity bursts that make statistical evaluation challenging16,17.
To better understand the molecular mechanosensitivity, we have developed two additional systems to examine TRPV4. (1) To isolate TRPV4 away from its usual mammalian partners, we have expressed rat TRPV4 in budding yeast24. Functional expression of TRPV4 in this evolutionarily distant context showed that it could still respond to osmotic force without its usual partners. Because yeast makes no PUFAs such as AA or EET, and its genome has no PLA2 or epoxygenase genes, this expression also shows that they are not required for TRPV4 to sense force. Having TRPV4 in the molecular biologically most amenable eukaryotes also allows efficient forward- or reverse-genetic manipulations25. (2) For in-depth biophysical analyses of TRPV4, we expressed TRPV4 in Xenopus oocytes. Unlike cultured cells, which yield sub-nA to nA (10-10 to 10-9 A) currents, an oocyte expresses currents in &#181;A&#39;s (10-6 to 10-4 A). Much larger signal over noise allows better quantification and more confident comparison. TRPV4, so expressed, can also be examined as individual molecules using a patch clamp. A single oocyte allows repeated patch sampling, again making quantification more reliable. Such studies showed that TRPV4 channel itself can directly be activated by membrane stretch force14. Analyses also showed that 14 representative skeletal-dysplasia alleles are all gain-of-function mutations. Further, the degree of this constitutive Ca2+ leakage parallels the severity of the skeletal diseases23.
Because of their novelty and usefulness, the detailed procedures of these two methods are assembled here to allow replications in future research on TRPV4 or similar channels.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

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 Ca2+, 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.

In a typical luminometry experiment, a range of 400-1,200 mOsM hypotonic down shocks is achieved by adding 200 &#956;l of shock solutions of different low osmolarity to 20 &#956;l of culture at 1,400 mOsM. The TRPV4 activity is evident when the RLU of the experimental is compared to that of two negative controls: yeast cells transformed with empty p416GPDV4 plasmid or one that contains the TRPV4 gene with a mutation at the ion filter (Figure 1)24.
In a typi...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

10.0K 조회수.  University of Wisconsin – Madison.  TRPV4의 연구에 일반적으로 사용되는 방법을 보완하기 위해 두 가지 방법이 설명되어 있습니다 : 그것의 mechanosensitivity는 저자 삼투 도전에 따라 형질 전환 효모에서 칼슘 2 + - 애큐 오린 luminometry으로 공부하실 수 있습니다. 그것은 또한 TRPV4-RNA에서 조사 할 수있는 온 - 셀 또는 절제 모드 클램프 전체 셀 두 전극 전압 클램프 또는 패치로 Xenopus의 난?...