A single molecule study on the structural basis of ion selective permeation in voltage-gated sodium channels

High resolution studies of voltage-gated sodium channels (VGSCs) can not reveal the dynamic nature of the channel structures, especially in lipid environments, which are vital for rational drug design. The bacterial NavAb channel is highly like its eukaryotic orthologs in structure, function, and pharmacological profiles, serving as an ideal model for biochemical characterizations. I purified the NavAb channel proteins and reconstituted them into liposomes. With liposome flux assays, I showed that the purified NavAb channels are highly conductive for Na+ and H+, but less permeable to K+, Cs+, and the bulky cation N-methyl-D-Glucamine. The NavAb channel is cysteine free, which allowed for introduction of cysteine mutations at different sites to be labeled with fluorophores. Thus, the structural dynamics of the NavAb channel can be examined utilizing single-molecule FRET. It is proposed that the ion selectivity of VGSCs is based on the size and charge, implying very minimized flexibilities in the selectivity filter. However, it was found that selectivity collapse underlies the slow inactivation in VGSC, including NavAb. To address the discrepancies, I examined the structural dynamics of the NavAb selectivity filter under different ionic conditions and membrane voltages. Surprisingly, my smFRET data showed large spontaneous FRET transitions which suggest significant structural dynamics at the NavAb selectivity filter. Moreover, I also detected significant FRET changes at the NavAb selectivity filter induced by activating voltage. My results indicate that ion selectivity in VGSC is not completely based on size and charge and needs to be investigated further.

Name thesis:degree_name

M.S. (Master of Science)

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Cellular and Molecular Biology (UMKC)

Grantor

University of Missouri--Kansas City

Year dc:date.issued

2021

Author dc:creator

• Vance, Joshua

Advisor dc:contributor.advisor

• Wang, Shizhen

Handle dc:identifier.uri

https://hdl.handle.net/10355/86702

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/86702