Dissertations, Theses, and Capstone Projects

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Mitchell Goldfarb

Committee Members

Jayne Raper

Paul Feinstein

Sebastien Poget

Jianmin Cui

Subject Categories

Molecular and Cellular Neuroscience | Molecular Biology | Structural Biology

Abstract

During an action potential, voltage-gated sodium channels (VGSC) control sodium ion flow into the cytoplasm of cells by using membrane voltage to alter channel conformation. These membrane proteins exhibit three main functional conformations: closed, open, and fast-inactivated, with transitions between these states dependent on membrane voltage and kinetics. The channels open within 1 millisecond and swiftly transition from open to fast-inactivated within 1-2 milliseconds following membrane depolarization. Subsequent membrane repolarization resets the channels to the closed state. Notably, even at mildly depolarized potentials that preclude channel opening, VGSCs can undergo transitions between the closed and inactivated states, known as closed-state inactivation, which equilibrates to steady-state inactivation within 60 milliseconds. The percentage of channels available for conducting sodium current is influenced by closed-state inactivation, as channels can only transition to the open state from the closed state. Furthermore, the likelihood of inactivation from the closed state increases as the membrane voltage approaches the threshold for channel opening.

Although earlier studies and recent cryogenic electron microscopy structures have shed light on the mechanisms by which membrane voltage controls the closed-to-open state transition, the structural mechanisms that govern the voltage setpoint for channel inactivation remain unknown. Guided by structures of VGSCs and that of its C-Terminal Domain (CTD) fragment in complex with Fibroblast Growth Factor Homologous Factor – 2 (FHF2), we hypothesized that a VGSC intramolecular interaction between the cytoplasmic Domain III-Domain IV (DIII-DIV) inactivation loop and the CTD acts as a mild restraint against channel inactivation as membrane voltage rises. Furthermore, FHF binding to the CTD might add additional contacts between the CTD and DIII-DIV loop, requiring greater depolarization for disengagement and channel inactivation.

Amino acid substitutions targeting residues at the predicted CTD – DIII-DIV interface were engineered into Nav1.5 plasmids that were then expressed in Neuro-2a cells. Using the whole-cell voltage-clamp technique, we observed that these substitutions hyperpolarized the voltage dependency of inactivation. We also observed that hyperpolarizing shifts in the V1/2 of inactivation caused by single amino acid substitutions were less than that caused by deleting the entire CTD. We further investigated FHF modulation of VGSC inactivation by making amino acid substitutions into Nav1.5 and Nav1.6 plasmids targeting residues within the juxtamembrane (JM) region suspected of making hydrophobic contacts with the III-IV loop upon FHF2 binding. Patch clamp analysis after co-expression of these JM mutant VGSCs with FHF2 showed that the JM mutants had a similar V1/2 of inactivation to their respective wild types in the absence of FHF2. However, when FHF2 was co-expressed with these JM mutant VGSCs, FHF2 was unable to depolarize the V1/2 of inactivation as effectively as it could the Nav1.5 WT and Nav1.6 WT channels. Additionally, the epileptogenic FHF1B R52H gain-of-function mutant was also less effective in right-shifting the V1/2 of inactivation of the Nav1.5 JM mutant as much as it did the Nav1.5 WT. These data suggest a generalized mechanism by which different FHF isoforms modulate different isoforms of VGSCs.

Understanding these underlying structural mechanisms furthers our understanding of the intrinsic and modulatory mechanisms governing the voltage set points of VGSC inactivation. This understanding may help in the design of drugs to treat channelopathies like Long QT 3 (LQT3) syndrome and congenital insensitivity to pain, diseases associated with VGSC inactivation gating. Additionally, mutations in FHFs can also cause devastating diseases such as Early Infantile Epileptic Encephalopathy-47, a disease that is refractory to anti-epileptic drugs, and thus this study may also assist towards designing treatments for such clinical diseases.

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