Voltage-gated sodium channels auxiliary subunits evolutionary emerged nearly 500 million years ago during the Cambrian explosion. These subunits alter one the most important ion channels to electrical signaling, the voltage-gated sodium channels support the propagation of electric impulses in animals. The mechanism for the auxiliary subunits effects on the channels is poorly understand, as is the stoichiometry between the auxiliary subunit and the channel. The focus of my thesis is to generate assays and to use these approaches to understand the interactions different types of voltage-gated channels and their auxiliary subunits. A biochemical approach was taken to identify novel interactions between the eukaryotic sodium channel auxiliary subunits and a prokaryotic voltage-gated sodium channel, a protein that diverged from the eukaryotic voltage-gated sodium channels billions of years ago. These interactions between the auxiliary subunits and channels were probed with chemical and photochemical crosslinkers in search of interaction surfaces and similarity to explain the mechanisms of interaction. The work in this thesis identified novel interactions between the voltage-gated sodium channel auxiliary subunits and voltage-gated channels that are distantly related to the voltage-gated sodium channels principally thought to be modulated by the auxiliary subunits. From this work a rudimentary concept can be theorized that the voltage-gated sodium channel β-subunits and not only β1 have a more primary role in electrophysiology by associating with multiple different types of ion channels.

Indiana University-Purdue University Indianapolis (IUPUI) / Increased electrical activity in peripheral sensory neurons contributes to pain. A unique type of sodium current, fast resurgent current, is proposed to increase nerve activity and has been associated with pain pathologies. While sodium channel isoform Nav1.6 has been identified as the main carrier of fast resurgent currents, our understanding of how resurgent currents are modulated in sensory neurons is fairly limited. Thus the goal of this dissertation was to identify resurgent current modulators. In particular, we focused on sodium channel beta subunits (Navβs) and fibroblast growth factor homologous factors (FHFs) in dorsal root ganglion (DRG) neurons. We hypothesized that Navβ4 and FHF2B act as positive regulators by mediating resurgent currents and modulating Nav1.6 inactivation, respectively. In contrast, we hypothesized FHF2A negatively regulates resurgent current by increasing the probability of channels in inactivated states. Thus, the aims of this dissertation were to 1) determine if Navβ4 regulates fast resurgent currents in DRG neurons, 2) examine the effects of Navβ4 knockdown on resurgent currents, firing frequency and pain associated behavior in an inflammatory pain model and 3) determine if FHF2A and FHF2B functionally regulate Nav1.6 currents, including resurgent currents in DRG neurons. To examine the aims, we used biochemical, electrophysiological and behavioral assays. Our results suggest that Navβ4 is a positive regulator of resurgent currents: in particular, the C-terminus likely mediates these currents. Localized knockdown of Navβ4 decreased inflammation-induced enhancement of resurgent currents and neuronal excitability, and prevented the development of persistent pain associated behavior in an inflammatory pain model. FHF2B increased resurgent currents and delayed inactivation. In contrast, FHF2A limited resurgent currents; an effect that is mainly contributed by FHF2A's N-terminus activity that increased accumulation of channels in inactivated states. Interestingly, in an inflammatory pain model FHF2B was upregulated and FHFA isoforms were downregulated. Together these results suggest that FHF2A/B modulation might contribute to enhanced resurgent currents and increased neuronal excitability observed in the inflammatory pain model. Overall, our work has identified three resurgent current modulators FHF2A, FHF2B and Navβ4. Manipulation of these proteins or their activity might result in novel strategies for the study and treatment of pain.

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