Exploring the Role of Pore Waters and Counterions in the Calcium Release-Activated Calcium Channel Conductance with Computation

1602-Pos Board B332 Mechanism of Activation of Calcium Channel Orai1 by its Regulatory Partner Stim1 Aparna Gudlur, Ariel Quintana, Yubin Zhou, Anjana Rao, Patrick G. Hogan. La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA. Store-operated calcium entry in T lymphocytes depends on the sensing by STIM1 of cellular calcium store depletion, leading to an interaction of STIM1 with ORAI1 and culminating in calcium flux through the ORAI1 channel. Some important features of the STIM1-ORAI1 interaction have been inferred from studies of mutated channels. A recently published structure of the Drosophila Orai channel, which has high sequence homology with human ORAI1, revealed the resting state pore architecture of the channel and confirmed evidence from biochemical and electrophysiological studies that transmembrane helices 1 line the pore. However, the critical steps of STIM1 binding and the resulting transition of the channel to its active calciumconducting state are yet to be delineated. Here we demonstrate a STIM1mediated conformational change in the purified wildtype ORAI1 channel that correlates with calcium flux through the channel reconstituted into liposomes. Introduction of the known disabling mutation R91W blocks the conformational change as well as calcium flux through the reconstituted channel. Mutations in the N-terminal cytoplasmic region of ORAI1 that disrupt STIM1 interaction with this part of ORAI1 are sufficient to abrogate the gating signal, although these mutations do not affect STIM1 interaction with the C-terminal region of ORAI1. Our study offers key insights into the STIM1-dependent gating of the ORAI1 channel.