Open Collections

Russo, Sophia Carolina

University of British Columbia

Background: Mutations of the KCNQ1 cardiac ion channel are known causes of arrhythmia disorders, including LQT-interval syndrome. This unique channel deviates from the conventional mechanism driving voltage-gated ion channel opening, as channel conductance is observed in mutants holding the VSD in both the expected fully-activated conformation and an elusive intermediate orientation. The objective of this study was to utilize a mutant known to produce currents characteristic of this intermediate-open state, E1R/R2E, to recapitulate the structure of the channel in this conformation. Methodology: Extensive troubleshooting was done to isolate protein of adequate yield and purity. Avenues for optimization included cell culture technique, choice of detergent and membrane isolation. Successfully isolated protein was vitrified, screened and finally assayed for data collection. Movies were then put through a cryoSPARC processing pathway with a model built using Coot from a KCNQ1 wild-type starting model. Phenix was used for final refinements. Results: A 3.66 Å data set was successfully determined using cryo-EM, with the 3D model showing a closed pore and VSD distinct from wild-type. Though the resolution in the VSD is adequate for main chain conformation, it is not adequate to confidently make assertions about residue interactions. Interestingly, it does not appear that the expected salt-bridge interaction believed to stabilize the intermediate state in this mutant is possible, given the model. Major observation included a slight downward movement of the S4 and significant movement of the S1 and S2 helices in the VSD. Conclusion: This model is the first-ever high-resolution structural model of the KCNQ1-R2/CaM and therefore fills an important knowledge gap about the intermediate state.

Text

2024-10-24

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89500

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/