TRPY1, a TRP Family Channel in Yeast

There is a prominent inwardly rectifying, 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).

Like other TRP channels, TRPY1 can be activated by several means.  Its behavior in different  [Ca²⁺] and applied pressures indicates that it sums the two energies, as if they act in parallel.  To map this formalism to structure, we found calmodulin or calcineurin to be unnecessary.  Removing a dense cluster of negative charges in the C-terminal cytoplasmic domain, however, greatly diminishes the  Ca²⁺ activation as well as its influence on force activation.  Thus, it appears that  [Ca²⁺ ] binding to the cytoplsmic domain and stretch in the membrane-embedded domain both generate gating force, reaching the gate in parallel (6).  See Fig. 3.

Fig. 3.  Stretch force and cytoplasmic Ca2+ activate the gate of TRPY1 in parallel.  Diagram based on patch-clamp data as well as the use of a calmodulin mutant and several engineered mutations in TRPY1 (6).

Taking advantage of the advanced molecular genetics of the yeast system, we have isolated many gain-of-function mutants of TRPY1.   See "Genetics" below.  For a phenotype, we follow, by luminometry, the hypertonically induced rise of [Ca²⁺] with transgenic aequorin.  Gain-of-function (GOF) mutants were isolated that gave much larger response to even mild hypertonic shocks.  Surprisingly, many of these GOFs have mutations in aromatic amino acids.  In one case, changing the identified Y458 residue to all 19 other possibilities showed that only a Y458F and Y458W retain normal gating, indicating the importance of aromaticity (7).  Although aromatics serve many functions, one possibility is that they stabilize the channel in the lipid bilayer at the polar-nonpolar interface.  We tested aromatic compounds and found that indole and other aromatic compounds added to cells in vivo or to membranes under patch clamp clearly activate TRPY1.  We speculate that these compounds alter the innate forces in the bilayer received by the channel (8).

TRPY1 gain-of-function mutations cause severe aberrations in channel kinetics and open probability.  They are found to be located at the two ends of S6 and its immediate C-terminal extension as well as the base of S5 near S4.  (The S4-S5 linker is known to operate the S6 gate in voltage-gated channels.)  Here we found the TRPY1 GOF mutation, F380L, in a cluster of phenylalanines, members of which can be found in all TRP subfamilies (9).  Curiously, a unique mutation at the same phenylalanine (F550I) in TRPC1 caused a GOF phenotype in Drosophila retina.  More recently, a screen of GOF in rat TRPV1 in yeast also yielded a mutation at the same position.  Further, the varitint-waddler mouse TRPML1 GOF mutation and the human TRPV4 GOF mutations, which cause autosomal-dominant brachyolmia, are all found to be nearby.  All five cases are results from assumption-free forward-genetic searches.  The same molecular phenotype (constitutive currents) and their coincidence strongly suggest a commonality of a gating mechanism across all TRP subtypes that uses the base of S5, likely the aromatic residues therein (10).  See Fig. 4.

Fig. 4. Fig. 1  An alignment of the S5's (the predicted 5th transmembrane a helices) of different TRP subtypes showing coincidence of gain-of-function mutations.   In three independent unbiased searches, the same site (red star) was discovered, at which mutations cause constitutive channel activities in Drosophila TRPC1, rat TRPV1, and yeast TRPY1 (bold red letters).  The site is in a small cluster of phenylalanines, members of which are found in all TRP subtypes (underlined red).  The mutations in TRPV4 that causes brachyolmia in human (green) and the mutation in TRPML that causes the varitint-waddler mouse phenotype are nearby (orange).  Shown are subfamily representatives: TRPA (painless of Drosophila), TRPC (the canonical TRPC of Drosophila), TRPM2 (human), TRPML3 (mouse), TRPN (zebra fish), TRPP2 (mouse), TRPV1 (rat), TRPV4 (rat) and TRPY1 (budding yeast).

 

1.	Palmer et al. (2001) PNAS 98: 7801.
2.	Denis & Cyert (2002) J. Cell Biol. 156: 28.
3.	Zhou et al. (2003) PNAS 100: 7105.
4.	Zhou et al. (2005) Eur. Biophys. J. 34: 413.
5.	Kung (2005) Nature 436: 647.
6.	Su et al. (2009) J. Memb. Biol. 227: 141.
7.	Zhou et al. (2007) PNAS 104: 15555.
8.	Haynes et al. (2008) FEBS lett. 582: 1514.
9.	Su et al. (2007) PNAS 104: 19607.
10.	Myers et al. (2008) J. Gen. Phy. 132: 481.

 

 

 

 

 

 

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