Animal TRPV4

As reviewed elsewhere (1,2,3), TRP homologs are found in many cellular eukaryotes including yeasts and other fungi, even though research tends to concentrate on the seven subtypes of animal TRPs.  Besides studying the TRP channel (TRPY1) native of S. cerevisiae (above) and related fungi (4), we have begun examining animal TRP channels expressed in yeast.  In parallel, the Julius lab has expressed TRPV1 in yeast and found GOF mutations therein.

Among the TRPV (vanilloid) subtype, TRPV1 is best known to respond to noxious heat and its surrogate, capsaicin, and TRPV4 to hypotonic shocks.  All TRPs are promiscuous and can be activated by multiple means.  Rat TRPV4 expressed in cultured mammalian cells can be activated by polyunsaturated fatty acids (PUFAs), phorbal esters, warm temperatures, and hypotonic shock.  Placing a mammalian TRP channel in cultured mammalian cells provides it with a similar environment, likely preserving much of its physiology.  However, such an environment is complicated, presumably having the channel in a complex network of native regulators, potentially making it difficult to tease apart the precise molecular mechanism by which specific stimuli lead to channel opening.  This is particularly difficult for stimuli such as temperature, which has profound global effects on cell metabolism.  Likewise, osmotic swelling over minutes globally induces many processes, making it difficult to know whether a TRP activation is due directly to membrane swelling itself or by a downstream consequence of swelling.  Indeed, there is evidence that swelling activates enzyme(s) that produce PUFAs to activate TRPV4 in HEK cells.  We expressed rat TRPV1 and TRPV4 in yeast where the rat channels are much less likely to be affected by multiple regulatory network as in animal cells. Thus, yeast expression may make it easier to fathom the channels' own molecular properties.

We used transgenic aequorin to monitor the cytoplasmic [Ca²⁺] in rat-TRPV4 expressing yeast cells in vivo (Fig. 1A) (5).  Hypotonic shock induces a rise in [Ca²⁺] in such yeast cells as in cultured mammalian cells (Fig. 1B) with response proportional to stimulus (Fig.1C).  Yeast cells bearing empty plasmids or TRPV4 gene with an occluding mutation at the ion filter (M680K) do not respond to the shocks (Fig. 1B,C).  We have also expressed rat TRPV1 in yeast.  Yeast expression apparently preserves the molecular characteristics of the animal channels expressed:  TRPV1 is activated by capsaicin but not by hypotonic shock; TRPV4 is activated by hypotonic shock but not capsaicin (Fig. 2).  Likewise, heat activates rat-TRPV1 but not TRPV4 in yeast (Fig. 3).  Like in some expressions in mammalian cells, expression of foreign channels can encounter traffic problems.  Rat-TRPV4 is apparently expressed in internal membranes of yeast, complicating direct patch-clamp analysis (5).

Fig. 1.  Rat TRPV4 expressed in yeast respond to hypotonic shock. (A) A diagram showing the experimental methods, modified from [11].  Yeast cells (in yvc1D background) were first transformed with a plasmid that produces aequorin to monitor cytoplasmic Ca2+ by luminescence.  They were then transformed with a CEN plasmid bearing the rat TRPV4 gene.  The transformed yeast culture was monitored with a luminometer and was hypotonically shocked by dilution. (B) A 750 mOsM hypotonic shock (arrow heads) triggers a large luminescence increase (in relative luminescence units, RLU) in TRPV4 transformants, but not in transformants of an empty plasmid, or plasmid bearing a TRPV4 with a mutation in its ion filter (M680K).  All shocks were in the presence of 25 mM EGTA.  Cells in post-logarithmic phase of growth.  (C) A dose-response relation between hypotonic shock and the peak response (Mean + S.D., n = 3).  Measurements from 2.4 x 106 cells each.

Fig. 2  The responses of TRPV1 and TRPV4 to capsaicin and to hypotonic shock.  (A) 10 mM capsaicin activates TRPV1 yeast tranformants (black).  This signal is completely removed by the addition of 25 mM EGTA (red).  The presence of 10 mM ruthenium red (green) also blocked the response completely.   TRPV4 transformants show no response to capsaicin (blue).  0.5 x 106 cells per test.  (B)  750-mOsM shock activates post-logarithmic transformants of TRPV4 (blue) but not TRPV1 (black) or empty plasmid (red). 10 mM ruthenium red (blue) has no effect on this response.    2.4 x 106 cells per test.

Fig, 3.  Noxious heat above 45o C causes a clear increase in cytoplasmic Ca2+ in TRPV1 transformants (upper three traces in each panel), whereas TRPV4 transformants (grey traces) do not have a response greater than yeast transformed with empty-plasmid (bottom black traces).  All cultures were in logarithmic phase of growth.  Heat is given as an estimate of the initial temperature at the point of injection , but little difference in the actual temperature between samples existed as evidenced by the consistency in responses, which are shown in triplicate. 1.2 x 106 cells per test.

The internal localization of TRPV4 made ineffective the external application of PUFAs, 4aPDD, and ruthenium red.   In want of pharmacological evidence, our claim that the Ca²⁺ luminometry response is indeed that of TRPV4 rests on two other types of evidence.  Physiologically, the robust response is to hypoosmolarity, resembling TRPV4's responses in live worm, in mammals, and in cultured mammalian cells.  Genetically, the response is absent with empty plasmid or when a point mutation is engineered at the filter of TRPV4.

PUFAs are needed for worm olfactory and nociceptive signaling with Osm-9, a TRPV homolog.  The specific PUFA, 5',6'-epoxyeicosatrienoic acid (5',6'-EET), not its isomers, is needed in hypotonic but not chemical or heat activation of TRPV4 in HEK cells.  It was proposed that hypotonic swelling activates phospholipase A2, releasing 5',6'-EET to activate TRPV4. The Saccharomyces cerevisiae genome was sequenced in 1996, first among eukaryotes. It has only a single fatty-acid desaturase, Ole1p, which produces Δ9 monounsaturated fatty acids. Yeast can take in polyunsaturated phospholipids from the medium, but the cells were cultured without lipid supplement here.  Yeast also could not have made PUFAs from such lipids, even if supplied, since it also lacks phopholipase A2 for the necessary hydrolysis.  Our findings therefore show that hypotonic shocks can activate TRPV4 without PUFAs, including 5',6'-EET in some context.

It is formally possible that hypotonic swelling produces an element in yeast that activates TRPV4 in place of PUFA.  However, it seems unlikely that yeast has an element that can fulfill the stringent chemical specificity of 5',6' EET.  It is also possible that PUFA relieves TRPV4 from an innate inhibition in the mammalian membrane, which is absent in the yeast membrane.  Yet another possibility is that TRPV4 itself senses the forces from the membrane.  PUFAs have been shown to change the internal force profile of lipid bilayer in molecular dynamics simulation.  It has also been proposed that PUFAs enter the two monolayers differentially straining the bilayer to activate TREK1, the mechanosensitive two-pore-domain K+ channel, as in the activation of MscL by amphipaths.  The yeast membrane has a different composition and may have an internal force profile that can activate TRPV4 without the need of PUFA addition.  Different types of lipid rafts may exist in different cells.  Direct patch-clamping offers best hope in understanding the molecular behavior of rat TRPV4.  We are attempting patch-clamp the yeast vacuolar membrane, one of the internal membranes of yeast, in search of rat-TRPV4 activities.  We also encourage animal physiologists to re-examine TRPV4 in culture mammalian cells at the physiological temperature of the rat (37^oC) instead of the more convenient room temperatures (~25^oC).

1.  Saimi et al. (2007) in Mechanosensitive Ion Channels, ed. O. Hamill,  pp.311-327.
2.  Kung et al. (2007) in Sensing with Ion channels, ed. B. Martinac, pp 1-23.
3.  Martinac et al. (2008) Ion channels in microbes. Physiol. Rev. 88: 1449-1490.
4.  Zhou et al. (2005) Eu. Biophys. J. 34: 413-422.
5.  Loukin et al. (2009) FEBS Lett. 583: 754-758.

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