Calcium-regulated ion channels control essential processes in nerves and muscles. Channelopathies associated with the voltage-dependent sodium channel family (denoted NaV1.n, VDSC or VGSC) include several severe forms of epilepsy, cardiac """"""""Long QT"""""""" syndrome 3 and ventricular fibrillation, familial autism, pain insensitivity, and other defects in the generation and propagation of action potentials. Most members of the NaV family of channels respond to calcium via regulation by calmodulin (CaM), an essential eukaryotic calcium sensor that is comprised of two highly homologous domains (N and C) connected by a regulatory linker. New solution structures show how apo (calcium-free) C binds to an IQ motif (IQxxx[R,K]Gxxx[R,K]) in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 (2KXW) and NaV1.5 (2L53). For both, Ca2+ binding to CaM changes the conformation of C and lowers its affinity for the NaV IQ motif. Although these structures and thermodynamic studies show that N does not bind to the NaV IQ motif, genetic studies indicate that mutations in N affect NaV function. Thus, a significant gap in our understanding of this calcium- mediated switching process is how and where N associates with NaV. We hypothesize that CaM serves as a calcium-triggered channel organizer;the N and C domains of CaM bring together distinct parts of NaV to regulate conformation and conductivity. We propose that apo CaM is tethered by C at the IQ motif. When Ca2+ binds to CaM, N binds to its preferred site in NaV, while C either (a) opens and twists on the IQ motif, with the same orientation, or (b) releases and rebinds with opposite orientation. These orientations would restrict the NaV interfaces available to N in different ways. To determine how CaM translates changes in intracellular [Ca2+] into conformational work, and regulates multiple isoforms of NaV, we will determine the distinct roles of N and C by testing how they recognize naturally occurring variations in isoforms of human NaV and how disease-causing mutations in NaV uniquely affect binding of apo and calcium-saturated N and C. We propose three Aims focused on sodium channels found in the central nervous system (CNS).
The first Aim will determine how apo and Ca2+-saturated CaM C-domain binds disease-causing mutant IQ motifs of NaV.
The second Aim will determine the effect of CaM on interactions between NaV IQ motifs and NaV """"""""EF-Hands"""""""".
The third Aim will determine where apo and (Ca2+)2-CaM N-domain are binding to isoforms of NaV. Structures of CaM will be studied by crystallography, NMR, and CD, while dynamics and energetics from titrations will be determined with fluorescent biosensors encoding known and putative CaM-binding domains, NMR, and calcium titrations monitored by steady-state fluorescence. We will discover key macromolecular orientations between CaM and NaV, provide the first structure of an NaV tail fragment containing both the EF-hand and IQ motif, and indicate the CaM-mediated contribution to multiple disease-associated mutations related to epilepsy, or identified as candidates for autism-spectrum disorders.

Public Health Relevance

Human voltage-gated sodium channels in the NaV family are regulated by calmodulin (CaM) and responsible for the rising phase of action potentials in the central nervous system, heart and skeletal muscle. Mutations that affect calcium response, folding, temperature sensitivity and the overall length of the protein are known to cause epilepsy, cardiac defects, and are candidates for genes underlying autism and ALS (""""""""Lou Gehrig's Disease""""""""). Although de novo carriers may not have offspring, high-throughput screening has opened new opportunities to identify deleterious sequence changes in the CaM-binding regions. We will determine how CaM remodels the intracellular C-terminal tail of NaV to provide calcium-mediated switching. Discovering the regulatory role of each domain of CaM lays the groundwork for developing future therapies and will provide information regarding side-effects of medications taken for epilepsy and other neurological disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057001-09
Application #
8326878
Study Section
Special Emphasis Panel (ZRG1-BCMB-S (02))
Program Officer
Flicker, Paula F
Project Start
1998-09-01
Project End
2016-02-29
Budget Start
2012-05-01
Budget End
2013-02-28
Support Year
9
Fiscal Year
2012
Total Cost
$321,473
Indirect Cost
$106,092
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Isbell, Holly M; Kilpatrick, Adina M; Lin, Zesen et al. (2018) Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7. Biomol NMR Assign 12:283-289
Fowler, C Andrew; Núñez Hernandez, Maria F; O'Donnell, Susan E et al. (2017) Backbone and side-chain resonance assignments of (Ca2+)4-calmodulin bound to beta calcineurin A CaMBD peptide. Biomol NMR Assign 11:275-280
Mahling, Ryan; Kilpatrick, Adina M; Shea, Madeline A (2017) Backbone resonance assignments of complexes of human voltage-dependent sodium channel NaV1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin. Biomol NMR Assign 11:297-303
Hovey, Liam; Fowler, C Andrew; Mahling, Ryan et al. (2017) Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2. Biophys Chem 224:1-19
Feldkamp, Michael D; Gakhar, Lokesh; Pandey, Nisha et al. (2015) Opposing orientations of the anti-psychotic drug trifluoperazine selected by alternate conformations of M144 in calmodulin. Proteins 83:989-96
Wang, Xinxin; Boyken, Scott E; Hu, Jiancheng et al. (2014) Calmodulin and PI(3,4,5)P? cooperatively bind to the Itk pleckstrin homology domain to promote efficient calcium signaling and IL-17A production. Sci Signal 7:ra74
Newman, Rhonda A; Sorensen, Brenda R; Kilpatrick, Adina M et al. (2014) Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1). Biophys Chem 193-194:35-49
Evans, T Idil Apak; Hell, Johannes W; Shea, Madeline A (2011) Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2. Biophys Chem 159:172-87
O'Donnell, Susan E; Yu, Liping; Fowler, C Andrew et al. (2011) Recognition of ?-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles. Proteins 79:765-86
Feldkamp, Michael D; Yu, Liping; Shea, Madeline A (2011) Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel. Structure 19:733-47

Showing the most recent 10 out of 30 publications