TRP ion channels play important roles as environmental sensors in many eukaryotic cells. TRP channel dysfunction is known to cause several specific diseases, and increasingly has been linked to a growing number of multifactorial illnesses. Understanding precisely how these channels are regulated in their cellular environment is critical to understanding the basic mechanism underlying TRP channel pathologies. This study uses the Yvc1p channel from the baker's yeast Saccharomyces cerevisiae as a model system to study channel regulation, focusing on the effect of the C-terminal domain. First, the membrane topology of the C-terminal domain will be established in vivo using a set of topology reporters. In particular, glycosylation and protease susceptibility consensus sites will be introduced at specific positions, and biochemical tests of the resulting channels will determine if the introduced segments are located in the cytoplasm or the vacuolar lumen. Next, the role of the C-terminal coiled-coil domain will be examined. Parallel biochemical studies of a soluble C-terminal coiled-coil domain and immunofluorescence studies of the full-length channel will detail the effect of the coiled-coil on channel trafficking. In vivo protease studies will be used to examine the functional consequences of removing the coiled-coil domain from fully assembled and trafficked mature channels. Finally, the functional importance of specific loops within the C-terminus will be established by changing the length of the linkers between structural elements, and through the use of split constructs expressed as two protein fragments. The broad goal of this work is to clarify molecular mechanism(s) by which C-terminal domains modulate TRP channel activity. These results of these studies will provide a basic foundation for the development of novel pharmacological interventions targeting this important class of channels.
TRP ion channels play critical roles as environmental sensors, and have been implicated in diseases ranging from polycystic kidney disease to bipolar disorder. This work will use a model TRP channel from the baker's yeast Saccharomyces cerevisiae to gain insight into how TRP channels are regulated by intracellular domains. These results of these studies will provide a basic foundation for the development of novel pharmacological interventions targeting this important class of channels.
