Abstract
Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+–CaM required the cytoplasmic amino acid stretch E33–Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+–CaM.
Highlights
Zebrafish and is proposed to be a mechanosensing channel
Ca2+ was suggested to interact with the negative charge cluster D573DDD576 within the C terminus of TRP yeast channel 1 (TRPY1) assumed to be exposed to the cytoplasm [17]
In the mutant yeast calmodulin (Cmd1)–6, the aspartate and glutamate residues at the first and 12th position of the three Ca2+-binding loops were replaced by alanine residues
Summary
The yeast vacuolar TRP channel (TRPY1, Yvc; hydrophilicity plot) (Fig. 1A) is activated by cytoplasmic Ca2+ [2, 5, 13, 14]. The negative charge cluster D573DDD576 involved in Ca2+ binding of the 25-mer peptide is buried in protein domains present in the C terminus of the 137 amino acids (GST-TRPY1-C [538–675]) and could not be accessed by Ca2+. Cmd but not Cmd significantly bound to both protein fragments of TRPY1 but only in the presence of Ca2+ (Fig. 1D). To confirm the latter interactions, we produced TRPY1-N and TRPY1-C with additional tags. Application of 1 mM Ca2+ or 1 mM Ba2+ from the cytosolic side induced inward and outward currents, which were significantly increased in cmd compared with wt vacuoles (Fig. 2, E, F, and H and Fig. S1, A–C).
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