Identification of Inhibitory Ca²⁺ Binding Sites in the Upper Vestibule of the Yeast Vacuolar TRP Channel

By vacuolar patch-clamp and Ca²⁺ imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca²⁺ and inhibited by Ca²⁺ from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca²⁺ (Hill coefficient, 1.5), suggesting that it may accommodate a Ca²⁺ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of ⁴⁵Ca²⁺. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca²⁺ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca²⁺ binding.