Dissertations, Theses, and Capstone Projects

Date of Degree

9-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Mitchell Goldfarb

Committee Members

Paul Feinstein

Allyson Friedman

Mario Delmar

Theodore Cummins

Subject Categories

Biology | Molecular and Cellular Neuroscience | Neuroscience and Neurobiology

Keywords

FHF2, FGF13, sodium channels, voltage-gated, inactivation, excitability

Abstract

Purpose: Fibroblast Growth Factor Homologous Factors (FHFs) are a group of proteins known to associate with and modulate voltage-gated sodium channels (Nav) in excitable cells. The four FHF genes are differentially expressed in specific cell-types, with FHF2 expressed prominently in the hippocampus, cerebral cortex, heart and dorsal root ganglia. Due to previous unavailability of an Fhf2 knockout mouse, this gene’s functions have been understudied in comparison to other those encoding other FHFs. The purpose of this research has been to better understand the normal physiological functions of FHF2 at the cellular and system levels in the heart, sensory nervous system and central nervous system. These studies also include functional analysis of a point mutation in FHF2 that causes early-onset epileptic encephalopathy (EOEE) in humans. Methods: A stable fertile Fhf2KO mouse line was derived and used for phenotypic observation, in vivo experimentation, tissue harvest and primary cell culture for imaging, protein analysis and electrophysiological recordings. Whole-cell patch clamp electrophysiology on primary cultured cardiomyocytes and dorsal root ganglion (DRG) cells supplemented with ectopic expression experiments in transfected HEK and Neuro2A cells was used to determine the modulatory effects of various FHF2 isoforms on various sodium channels. Results:FHF2 is required for proper cardiac function specifically under hyperthermic conditions. In the absence of FHF2, there is a breakdown in cardiac conduction due to the impairment of cardiomyocyte action potential generation. Without FHF2 there is a hyperpolarizing shift in cardiac Nav1.5 steady-state inactivation decreasing Nav1.5 availability. This reduces overall available sodium conductance at resting potential which, when paired with accelerated inactivation brought about by elevated temperatures, suppresses excitability. FHF2 is also necessary for topical heat nociception, as Fhf2KO mice do not respond to noxious heat. The nociceptive deficit is reproduced in mice where Fhf2 disruption is restricted to peripheral sensory neurons. Our experiments support an underlying mechanism that parallels the cardiac phenotype, as the absence of FHF2 induces hyperpolarizing shifts in the predominant sodium channel isoforms Nav1.7 and Nav1.8 expressed in DRG nociceptors. Reduced Nav1.7 availability is again exacerbated by more rapid channel inactivation at elevated temperature, which correlates with reversible temperature-sensitive conduction failure in Fhf2KO peripheral sensory fibers. Lastly, in light of recently discovered missense mutations in the A-type first exon of the FHF2 gene in several people suffering from EOEE, I have shown that these mutations prevent anti-excitatory FHF2A-mediated Nav1.6 fast-onset long-term inactivation without impairing the pro-excitatory modulation of Nav1.6 steady-state inactivation, likely leaving CNS neurons hyperexcitable and susceptible to the epileptogenic phenotype.

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