TRPY1 Rosetta-Based Model Gating (Quicktime movies)

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TRPY1, a TRP Family Channel in Yeast

Three groups of investigators independently encountered a prominent voltage-dependant, cation-selective, and Ca²⁺-activated ~300 pS conductance in the yeast vacuolar membrane, which can be directly examined with patch clamp (Fig. 1, left).

We found this channel to be the product of TRPY1 gene (formerly YVC1).  TRP (transient receptor potential) is a large superfamily of channels that are known to underlie animal’s sensations to certain chemicals, heat, cold, as well as mechanical stimulations. TRPY1 is the only gene in the yeast genome that predicts a TRP-channel subunit.  We found that deleting this gene removes the 300-pS conductance (1) (Fig. 1, right).

Fig. 1  (Left) A diagram showing the method of vacuole preparation and patch-clamping (1).  (Right) A pressure at 32 mm Hg activates the TRPY1 channel in the wild type(A), but not in the trpy1Δ (formerly yvc1Δ) vacuole even at a higher pressure and a higher [Ca2+] (B). Recorded in whole-vacuole  mode, (3)

Upon an osmotic upshock, the yeast vacuole shrinks and releases Ca²⁺ into the cytoplasm.  Denise & Cyert (2002) found this osmotically induced Ca²⁺ pulse missing in TRPY1-deleted mutant in vivo (2) (Fig. 3A, below).  Despite the fragility of isolated vacuoles, we have examined the effect of mechanical and osmotic pressure (Fig. 2) on them and discovered the TRPY1 is in fact mechanosensitive (3)

Fig. 2  Hyperosmotic shock activates TRPY1.  (A) A yeast vacuole under patch clamp equilibrated at 0.4 osmolar was subjected to an upshock by the addition of 1.5 osmolar solution (arrowhead) .  The vacuole shrank and showed channel activations within seconds.  (B) An amplitude histogram showing stepwise increment of the unitary currents in A .  From (3).

Upon an osmotic upshock, the yeast vacuole shrinks and releases Ca²⁺ into the cytoplasm.  Denise & Cyert (2002) found this osmotically induced Ca²⁺ pulse missing in TRPY1-deleted mutant in vivo (2) (Fig. 2A).  Despite the fragility of isolated vacuoles, we have examined the effect of mechanical (Fig. 3A) and osmotic pressure (Fig. 3B) on them and discovered the TRPY1 is in fact mechanosensitive (3)

Fig. 3  Phenotype in vivo.  Live yeast cells respond to osmotic upshock by releasing Ca2+ from the vacuole to the cytoplasm through TRPY1 channel.   Luminescence is registered indicating Ca2+ binding to aequorin expressed from a transgene in the cytoplasm (2).  (A) Rapid increase of luminescence is seen in wild-type S. cerevisiae cells upon an osmotic upshock by sorbitol addition. (B) Similar luminescence is observed in trpy1Δ cells expressing a TRPY2 transgene from K. lactis or TRPY3 from C. albicans.  From (4).

Deletion of TRPY1 resulted in no clear laboratory growth phenotype in various osmotic or other conditions.  Therefore, no attempts were made to find trpy1 loss-of-function mutants.  Random mutagenesis of plasmid-borne TRPY1 led to the isolation of mutant trpy1’s that hamper growth.  We found that the mutant proteins were not transported to the vacuolar membrane but were congested in the endoplasmic reticulum, interfering with the traffic of other proteins.  These mutants were not further investigated.
      Instead of growth inhibition, we have now used the Ca²⁺-induced luminescence triggered by a sub-threshold osmotic shock as the screening phenotype and have successfully isolated “gain-of-function” (“GOF”) mutants of TRPY1.  Seventeen such “GOF” mutants have been isolated.  Among these, we are currently most interested in those likely to have affected the mechanically openable gate.

TRPY1 of the budding yeast S. cerevisae has homologs in 18 other fungal genomes.  TRPY2 of Kluyveromyces lactis and TRPY3 from Candida albicans have been expressed in S. cerevisae and form functional  channels therein.  The mechanosensitivity of TRPY2 or TRPY3 can be demonstrated in vitro by patch clamp and in vivo through Ca²⁺ luminometry  (Fig. 3B).

See (5, 6, 7) for reviews on mechanosensitive TRP channels.

1.  Palmer et al. (2001) PNAS98: 7801.
2.  Denis & Cyert (2002) J. Cell Biol. 156: 28.
3.  Zhou et al. (2003) PNAS100: 7105.
4.  Zhou et al. (2005) Eur. Biophys. J.34: 413.
5.  Kung (2005) Nature436: 647.
6.  Saimi et al (2007) in Mechanosensitive Ion Channels, ed. O, Hamill,  Elsevier (in press)
7.  Kung et al. (2007) in Sensing with Ion Channels, ed. B. Martinac, Springer-Verlag (in press)

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