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 >....

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....

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