Exploring the STIM-Orai Interface in CRAC Channel Activation in Helminth Parasites

Store-operated Ca²⁺ entry signals are critical for cellular regulation mediated by the CRAC channel (Calcium-Release Activated Calcium Channel). This intricate signaling pathway involves the coupling of proteins in two different membranes: the STIM proteins, which are sensitive to Ca²⁺ and reside in the ER (endoplasmic reticulum); and the Orai channels of the PM (plasma membrane), which are highly selective for Ca²⁺. The molecular nature of the STIM-Orai coupling interface at the ER-PM junctions and the consequent activation of the Orai channel are processes that have been intensively studied, but have been little studied in the majority of parasitic helminths, which affect around one billion people worldwide, especially in developing countries, where sanitary conditions in many regions are precarious. In this project, we aim to study the gating mechanisms of CRAC channels from Schistosoma mansoni (Sm) and Ascaris lumbricoides (Al). It was observed that the expression of SmSTIM and SmORAI in HEK293 cells led to a constitutive Ca²⁺ entry, which was attributed to the C-terminal domain of SmSTIM. We will then investigate whether the C-terminal of A. lumbricoides will behave similarly throught the expression of a chimeric protein of SmSTIM with the C-terminus of A. lumbricoides. The chimeric protein was succefully cloned by Gibson assembly, as well as the full length AlSTIM, which will be subsequently expressed in mammalian cells to investigate its ability to function as a Ca²⁺ channel and the role of each domain in the gating mechanism A better understanding of the biophysical mechanisms of CRAC channels in helminths could significantly contribute to the development of new therapeutic strategies, improving public health worldwide.