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

Summary

To complement commonly used methods to study TRPV4’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.

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 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.

Introduction

TRPs (transient receptor potentials) comprise seven subfamilies of cation channels that serve sensory functions^1,2. 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 cloning^5-8. The latter method followed the gene product's response to hypo-osmolarity. TRPV4 is expressed in nearly all organs and functions in the development, physiology, or pathology of many disparate cell types^3,4.

Striking are the >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 P_Ca : P_Na ~7 ¹². Single-channel conductance rectifies at ~90 pS outward and ~40 pS inward^6,13,14. Heterologously expressed current (below) can be activated by hypo-osmotic swelling, shear stress, or warmth¹⁵. It is also activated by polyunsaturated fatty acids^16,17 and the synthetic phorbal ester 4αPDD 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 challenging^16,17.

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 µA'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.

Protocol

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 construction and transformation²⁷. Yeast strain and plasmids are available upon request.

1. Culture 2 ml of yeast cells overnight to post-logarithmic phase in a 30 °C shaker in leu- ura- "DCD" dropout medium²⁸ (a sulfate-depleted variant of the CMD-leu-ura medium²⁹), supplemented with 1M sorbitol. Inoculate 200 µl of these cultures into 1.8 ml of fresh medium supplemented with 2 µM of the luciferin coelenterazine. Grow in the dark at room temperature for another 24 hr without shaking.
2. Measure the osmolarity of the culture (typically at 1,400 mOsM) and other solutions (below) using a vapor pressure osmometer.
3. Aliquot 20 µl of fresh culture into a 12 mm luminometer tube for a single tube luminometer.
4. For the hypotonic shock, add 200 µl of a solution, containing 25 mM NaEGTA (pH 7.2) or NaMES (pH 7.2) and 500-100 mM NaCl, (total osmolarity of 1,200-400 mOsM) depending on the degree of shock desired. Continuously monitor the luminescence before and at least 120 sec after the osmotic downshock, interrupted only by the brief dilution operation. Register luminometer output on a desktop computer as relative luminescence units (RUL).

Though not for the study of transgenic TRPV4, we have used a variant of the above protocol in an automated system to examine the responses of a large number of yeast strains. Study of the responses to hypo-³⁰ or hyperosmotic³¹ stimuli of the yeast deletome (the collection of 4,906 yeast single-gene deletants) using a microplate luminometer and analyzed with corresponding software has been successful.

2. and 3. Oocyte Electrophysiology Methods

Electrophysiology uses basic methods³². PCR amplify the open-reading frame of wild-type or mutant TRPV4 using high-fidelity PfuUtra polymerase and integrated into pGH19 ³³. This entails a precise insertion of the ORF between the 5' and 3' UTRs of the Xenopus β-globin gene and downstream of a T7 RNA-polymerase promoter. Linearize the construct downstream of the 3' UTR and used as templates in an in vitro T7 RNA polymerase reaction. Use standard molecular-biological techniques³⁴.

Use stage V-VI oocytes from X. laevis. Animal husbandry, partial ovariectomy, defolliculation, and vitelline envelope removal follow standard procedures^32,33,35. Inject 2-40 ng of cRNA solution per oocyte using a Drummond Nanoinject II automatic microinjector. Inject ~30 oocytes for each set of experiments. After TRPV4-cRNA injection, incubate the oocytes in the ND96 medium with gentamicin (100 µg/ml) and 1 µM ruthenium red¹⁴.

2. Two Electrode Voltage Clamp

1. Pull borosilicate glass recording pipettes from precision disposable 100 µl micropipettes individually with a pipette puller.
2. Pull both voltage-measuring and current-injecting pipette electrodes are pulled to have a tip aperture of ~1 µm diameter.
3. Backfill electrodes with 2 M KCl resulting in 0.1-0.2 mΩ resistance. Mount them on HS2A headstages, which together with a VG-2Ax100 virtual-grounded bath clamp, are connected to a GeneClamp 500 amplifier interface through a Digidata 1440 digitizer. Acquire data using pClamp10 software.
4. Place the oocyte to be tested in a 1 ml bath in a fabricated plastic chamber mounted on the stage of a dissecting microscope with 20X magnification. The bath solution is virtual-grounded by a 3 M KCl agar bridge and a chlorinated silver wire placed close to the oocyte and connected to a VG-2A virtual ground stage.
5. Construct the chamber for continuous perfusion with plastic tubings as solution inlet and outlet. Connect the inlet tubing is connected to a fabricated perfusion system, which is a manifold of six 50 ml syringe shells, each filled with a different solution. Gravity feed rates of any particular solution is controlled to ~2-3 ml/min using "Keck" ramp clamps on the tubings.
6. Mount both electrodes with their headstages on micromanipulators. Perform electrode penetrations of the oocyte by micromanipulation.

3. Patch Clamp Examination of Direct Mechanosensitivity

The basic methods of patch clamp including pipette preparation, GΩ-seal formation, and formation of on-cell or excised modes are those in Hamill et al.³⁶ and Conn³².

1. Fill borosilicate glass pipettes with an ~1 µm diameter opening at the tip (bubble number 5-6) with 98 mM KCl, 1 mM MgCl₂, and 10 mM K⁺-HEPES, pH 7.2.
2. Attach the patch clamp pipette through a plastic tubing to a 5 ml syringe and through a T-joint, also to a manometer. Use a computer to handle both the electrode and the manometer outputs. Acquire data at 10 kHz, and then play back through an eight-pole Bessel filter at 1 kHz for analysis.
3. Different patches can be excised from the same oocyte repeatedly. This practice makes the examination of TRPV4's molecular activity more efficient and reliable.

Results

In a typical luminometry experiment, a range of 400-1,200 mOsM hypotonic down shocks is achieved by adding 200 μl of shock solutions of different low osmolarity to 20 μl 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 typical two-el...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vapor Pressure Osmometer	Wescor	Wescor 5500	Logan, UT, USA
Sirius Single Tube Luminometer	Titertek-Berhold	Sirius	Huntsville, LA, USA
Microplate luminometer	Titertek-Berthold	Mithras LB940	Huntsville, LA, USA
Luminometry software	Titertek-Berthold	MikroWin2000	Huntsville, LA, USA
Patch clamp and software	Axon Instrument	HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software	Union City, CA, USA
Manometer	Bio-Tec		Winooski, VT, USA
Pipette puller	Sutter Instrument Co	Model P-97	Novata, CA, USA
Microinjector	Drummond Scientific	Nanoinject II	Broomall, PA, USA
Luciferin coelenterazine	Invitrogen		Carlsbad, CA, USA
T7 RNA polymerase reaction	Ambion	mMessage mMachine	Austin, TX, USA
Gentamicin	Sigma
Ruthenium red	Sigma
Micropipettes	Drummond	100 microliter micropipets	Broomall, PA, USA
High-fidelity PfuUtra polymerase	Stratagene		La Jolla, CA, USA