Yeast Luminometric and <em>Xenopus</em> Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

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. Video LinkThe video component of this article can be found at

Volume sensing in the transient receptor potential vanilloid 4 ion channel is cell type–specific and mediated by an N-terminal volume-sensing domain

“…The volume-dependent activation of TRPV4 in Xenopus oocytes robustly occurred irrespective of PLA 2 activity (as tested with two different PLA 2 antagonists). This ability to sense volume changes in the absence of PLA 2 activity confirms previous studies on both oocytes and yeast (13,18), as well as on thoracic sensory neurons (31). Accordingly, whereas cell swelling may cause PLA 2 activation with subsequent accumulation of arachidonic acid and other metabolites (41,42,50), this enzymatic action seems to represent a required intermediary step for swellinginduced TRPV4 gating only in select cell types.…”

“…However, a key unresolved question pertains to the structural determinants that mediate the molecular coupling between cell swelling and channel activation. Müller glia, endothelial cells, and HEK293 cells may require an intermediate step involving phospholipase A 2 (PLA 2 ) activation and cytochrome P450 (CYP450)-dependent arachidonic acid (AA) metabolites for swelling-dependent activation of TRPV4 (1,8,17), whereas cell swelling has led to TRPV4 activation independently of PLA 2 and AA metabolites in retinal ganglion cells, sensory neurons, yeast, renal vascular cells, and Xenopus oocytes (1,13,18). It thus remains unresolved whether swelling-induced activation of TRPV4 can occur directly or whether an intracellular signaling cascade is required to couple cell swelling to TRPV4 activation.…”

A competing hydrophobic tug on L596 to the membrane core unlatches S4–S5 linker elbow from TRP helix and allows TRPV4 channel to open

“…Xenopus oocytes were injected with in vitro-synthesized TRPV4 cRNAs and recorded with a TEVC (two-electrode voltage clamp) after 3-4 d (14,27). Plasmid-borne TRPV4s were expressed and growth-phenotyped in yeast (14,28).…”

Mammalian TRP ion channels are insensitive to membrane stretch

“…In Drosophila, NOMPC is involved in many mechanosensitive processes, including gentle touch in larvae (Yan et al, 2013). In contrast, other TRP channels, such as TRPV4, clearly respond to mechanical force (Teng et al, 2013;Servin-Vences et al, 2017) but their mechanisms for doing so are subject of debate (Teng et al, 2013;Loukin et al, 2010). Although the deletion of ankyrin repeats in TRPV4 does not eliminate the mechanosensitivity of the channel, it does reduce its sensitivity to hypotonicity.…”