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).
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)
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.
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.
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