Abstract
SummaryBy 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.
Highlights
The transient receptor potential (TRP) ion channel family is encoded by more than 100 genes (Venkatachalam and Montell, 2007)
With the exception of the Ca2+-selective TRPV5 and TRPV6 the TRP channels are non-selective cation channels sharing the membrane topology of six transmembrane helices (S1–S6) and N- and C-terminal domains residing within the cytosol
Most TRP channels fulfill their physiological function in the plasma membrane as cation influx channels, whereas some functional TRP channels are localized in the membrane of cytoplasmic organelles (Berbey et al, 2009; Chen et al, 2014; Dong et al, 2008; Lange et al, 2009; Oancea et al, 2009; Turner et al, 2003)
Summary
The transient receptor potential (TRP) ion channel family is encoded by more than 100 genes (Venkatachalam and Montell, 2007). TRP channels shape the membrane potential and increase the cytosolic Ca2+ concentration ([Ca2+]cyt), translating environmental and endogenous stimuli into cellular signals. Likewise the TRP channel TRPY1, encoded by the vacuolar conductance 1 (YVC1) gene in Saccharomyces cerevisiae and localized in the vacuolar membrane, is activated by Ca2+ interfering with cytosolic channel domains (Bertl and Slayman, 1990; Palmer et al, 2001; Wada et al, 1987). The exact function of the TRPY1-mediated Ca2+ release is still elusive
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