Mechanisms of Channel Activation in GLIC

Abstract

GLIC is a pentameric proton-gated ion channel with an EC50 at pH of 5.0. It remains unclear which regions of GLIC are responsible for channel activation. In this study, we explore two potential regions by combining mutagenesis/functional experiments with computations using the Perturbation-based Markovian Transmission (PMT) model. The first region involves inter-subunit salt bridges between the C-loop (E177 and D178) and β5 (D91) of the complementary side. The second region (N152, D153, and D154) was part of the cavity identified crystallographically for anesthetic ketamine binding (Pan, Chen, et.al, Structure, 20, 1463, 2012). Mutations in the first region (D91N, E177Q, D178N) removed salt bridges and resulted in an increase of the EC50 (decreased pH to 4.8), suggesting the importance of the salt bridges in stabilizing the open-channel conformation, as confirmed by the less closed C-loop in the X-ray crystal structure. The mutation in the second region (N152C) resulted in a decreased of the EC50 (increased the pH to 5.4), presumably due to more flexibility for the protonation of D153 and D154. Using the PMT model, which provides time dependent information on the pathway of signal propagation, we found that perturbations to the first and second regions propagated through two different paths to the pore-lining TM2. The perturbation to the first region travels mainly from loop C, down β10 and β7 to the Cys-loop, and on to TM1 and TM3, which together affect TM2. The perturbation in the second region starts in the middle of the F-loop, travels down along the F-loop into the TM2-TM3 linker, and then to TM2. Taken together, our results reveal residues important to channel activation and allosteric signal pathways. Supported by NIH (R01GM066358, R01GM056257, R37GM049202, and T32GM075770).