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Xiao, Jenny

University of British Columbia

The human heartbeat is governed by a series of tightly controlled action potentials (APs) leading to the coordinated contractions of distinct groups of cardiac cells. Ion channel proteins are the cornerstones to this process, generating the different phases of the AP. Specifically, the cardiac voltage-gated sodium channel, Nav1.5, is responsible for generating the rapid depolarization phase of the AP. The channel possesses several domains and motifs which allow for an additional layer of control via cytosolic protein partners such as calmodulin (CaM) and fibroblast growth factor homologous factors (FHFs or FGF11-14). Dysregulation of either the channel or its protein partners can give rise to life-threatening arrhythmia syndromes. The goal of this thesis is to study the effects of calmodulin mutants, specifically F141L CaM on the structure and function of Nav1.5 and explore how FGFs engage Nav1.5. ITC data showed no significant difference between WT CaM and F141L CaM binding to the Nav1.5 CTD in either the presence or absence of Ca²⁺. The ternary structure complexes of the Nav1.5 CTD, WT apo/CaM, and FGF14 and Nav1.5 CTD, F141L apo/CaM, and FGF14 were determined at a resolution of 2.20Å and 1.60Å, respectively. The F141L mutation resulted in a destabilization of a hydrophobic pocket within the C-lobe causing a conformational change of a loop within EF-hand IV of CaM. The interface between FGF14 and Nav1.5 EF-hand domain revealed the absence of two salt bridges present at the interface of FGF13U and FGF12B with the Nav1.5 EF-hand supporting the observed decreased affinity. Moreover, ITCs showed that under Ca²⁺-saturating conditions, the change in enthalpy during titrations between FGF14 and CaM-bound Nav1.5 CTD was 2-fold lower than between FGF14 and Nav1.5 CTD. This points to differences in the binding of FGF14 in the Ca²⁺-saturated condition in support of a model where the Ca²⁺/C-lobe is bound to a position that would clash with a 180 rotation of the Nav1.5 EF-hand. ITCs between FGF14 and CaM-bound Nav1.5 CTD in the apo condition demonstrated a more complex two-site binding model suggesting the presence of two conformations of the apo/CaM-Nav1.5 CTD complex with which FGF14 can interact.

Text

2022-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/79076

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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