Dissertation
Recognition of NaV1.2 by calmodulin and fibroblast growth factor homologous factors
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005654
Abstract
The voltage-gated sodium channel 1.2 (NaV1.2) is a transmembrane protein primarily expressed in the central nervous system where it is responsible for generating the rising phase of an action potential. To do this, NaV1.2 undergoes voltage-dependent conformational changes that transition the pore of the channel from a closed to an open state, which allows Na+ to rapidly enter the neuron. Milliseconds after opening, the flow of Na+ through NaV1.2 is halted through a process known as inactivation. The ability of NaV1.2 to rapidly transition among closed, open and inactivated states is critical to its physiological function. Mutations found throughout the protein sequence of NaV1.2 that disrupt this process have been shown to cause debilitating disorders such as epilepsy and autism. Cycling of NaV1.2 among its functional states is tightly regulated by a network of interactions. These include intramolecular interactions between the cytosolic domains of the channel and intermolecular interactions between the cytosolic N- (NTD) and C-terminal domains (CTD) of the channel and several auxiliary proteins. Among these auxiliary proteins are fibroblast growth factor homologous factor 12 (FGF12) and the ubiquitously expressed Ca2+-sensor calmodulin (CaM).
FGF12 and CaM modulate NaV1.2 function through binding to the cytosolic CTD of the channel, with FGF12 binding to an EF-hand like domain (EFL), and CaM to an IQ motif (IQxxx[R,K]Gxxx[R,K]). However, how these two auxiliary proteins regulate NaV1.2 function is poorly understood. My thesis has focused on understanding the thermodynamic and structural mechanism through which CaM functions as a Ca2+ sensor when bound to NaV1.2. I have used spectroscopic methods to investigate the interaction between Ca2+-depleted (apo) and Ca2+-saturated CaM and the human NaV1.2 IQ motif as a peptide (NaV1.2IQp) and in a larger fragment of the NaV1.2 CTD containing both the EFL and IQ motif (NaV1.2CTD), and examined how pathogenic mutations in CaM perturb its interaction with the NaV1.2 IQ motif.
My solution structure of apo CaM bound to the NaV1.2IQp and other NMR studies of apo and Ca2+-saturated CaM bound to NaV1.2IQp and NaV1.2CTD have demonstrated that both endstates of CaM bind the NaV1.2IQp and NaV1.2CTD exclusively through the C-domain of CaM (CaMC), with the N-domain of CaM (CaMN) making no persistent contacts. Binding of NaV1.2IQp had markedly different effects on the residues in CaMC of apo and Ca2+-saturated CaM, and were consistent with the binding of Ca2+ inducing rotation of full-length CaM on both the NaV1.2IQp and NaV1.2CTD. This suggests that as the local Ca2+ concentration rises, CaMC may transiently release and re-associate with the NaV1.2 IQ motif. Pathogenic CaM mutants bound the NaV1.2 IQ motif as well as or with greater affinity than WT CaM under apo conditions. However, mutations in CaMC inhibited or ablated Ca2+ binding by CaMC when bound to the NaV1.2 IQ motif. Thus, CaM harboring these mutations would not be expected to support NaV1.2 regulation requiring rotation or transient release of CaM from the IQ motif.
CaM and FGF12 can interact simultaneously with the NaV1.2 CTD, placing these two auxiliary proteins near one another in the context of the channel. However, prior to this thesis no direct interaction had been reported between CaM and any FGF12 splice variant. My studies have discovered that both domains of CaM bind to a novel CaMBD in the NTD of the A-type splice variant of FGF12 (FGF12A CaMBD). CaM was found to bind the FGF12A CaMBD with high affinity in a Ca2+-dependent manner. Binding of full-length FGF12A to the NaV1.2CTD was found to increase the Ca2+ affinity of both CaM domains, consistent with Ca2+-saturated CaM interacting favorably with the NTD of full-length FGF12A when bound to NaV1.2CTD. This suggests that at a high cytosolic Ca2+ concentration, CaM may translocate from the NaV1.2 IQ motif to the FGF12A CaMBD to modulate NaV1.2 function. This provides new insights into the structural mechanism by which Ca2+, CaM, and FGF12A may act as regulators of NaV1.2.
Details
- Title: Subtitle
- Recognition of NaV1.2 by calmodulin and fibroblast growth factor homologous factors
- Creators
- Ryan Mahling
- Contributors
- Madeline A Shea (Advisor)Peter A Rubenstein (Committee Member)Ernesto J Fuentes (Committee Member)Catherine A Musselman (Committee Member)Adrian H Elcock (Committee Member)Janice L Robertson (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005654
- Publisher
- University of Iowa
- Number of pages
- xix, 444 pages
- Copyright
- Copyright 2020 Ryan Mahling
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 428-444).
- Public Abstract (ETD)
Much like circuits in a computer, nerves within the human body use electrical signals, known as action potentials, to communicate. Those signals initiate with a protein called the voltage-gated sodium channel 1.2 (NaV1.2). NaV1.2 senses changes in the voltage across the cellular membrane. A large change causes NaV1.2 to open, allowing sodium to enter the cell, resulting in the generation of an action potential. Within milliseconds after opening, NaV1.2 closes, halting the flow of sodium into the cell. The rapid closing of NaV1.2 is critical to its function within the neuron. Entry of excess sodium can cause uncontrolled generation of action potentials. This can cause debilitating seizure disorders such as epilepsy.
The opening and closing of NaV1.2 is tightly regulated by the binding of auxiliary proteins , including calmodulin (CaM) and the fibroblast growth factor homologous factor 12 (FGF12). However, the molecular mechanism by which CaM and FGF12 regulate the opening and closing of NaV1.2 is poorly understood. My research has focused on how CaM and FGF12 bind NaV1.2, and how the binding of CaM to NaV1.2 changes in response to calcium, another ion that controls cellular responses to electrical signals. The results of my studies suggest that, at an elevated calcium concentration within a cell, CaM may release NaV1.2 and bind FGF12A. This calcium-dependent switch in the location of CaM may contribute to the rapid closing of NaV1.2 necessary for synchronizing neuronal signaling
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984035694802771
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