Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2.
Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca2+)4-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMCbound to NaV1.2IQp. The polarity of (Ca2+)2-CaMCrelative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp(2KXW), revealing that CaMCrecognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMNallowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Liam
L
Hovey
NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 52242-1109 Iowa City, United States.
C Andrew
CA
Fowler
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Ryan
R
Mahling
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Zesen
Z
Lin
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Mark Stephen
MS
Miller
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Dagan C
DC
Marx
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Jesse B
JB
Yoder
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Elaine H
EH
Kim
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Kristin M
KM
Tefft
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Brett C
BC
Waite
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States.
Michael D
MD
Feldkamp
NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 52242-1109 Iowa City, United States.
Liping
L
Yu
Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United States. Electronic address: madeline-shea@uiowa.edu.
Madeline A
MA
Shea
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D051381:Rats
D002147:Calmodulin; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; D002118:Calcium
Allostery; Binding; Biosensor; FRET; Free energy; Linkage; Molecular recognition; NMR; Sodium channels; Thermodynamics; Titration