Solution Structure of the NaV1.2 C-terminal EF-hand Domain

doi:10.1074/jbc.M807401200

Solution Structure of the Na_V1.2 C-terminal EF-hand Domain^*

Departments of ^{^‡}Biochemistry and Molecular Biophysics and ^{^§}Pathology, Columbia University, New York, New York 10032, ^{^¶}Department of Biological Sciences, Columbia University, New York, New York 10027, and ^{^∥}Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, North Carolina 27710

4 A member of the New York Structural Biology Center (supported by National Institutes of Health Grant GM66354). To whom correspondence should be addressed: 701 West 168th St., Box 36, New York, NY 10032-3702. E-mail: agp6{at}columbia.edu; Tel.: 212-305-8675; Fax: 212-305-6949.

Abstract

Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human Na_V1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four α-helices separated by two short anti-parallel β-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the Na_V1.2 C-terminal domain does not bind Ca²⁺ through the canonical EF-hand loops, as evidenced by monitoring ¹H,¹⁵N chemical shifts during aCa²⁺ titration. Backbone chemical shift resonance assignments and Ca²⁺ titration also were performed for the Na_V1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca²⁺ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of Na_V1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the Na_V1.2 (1777-1882) structure.

Footnotes

↵⁵ The abbreviations used are: VSGC, voltage-gated sodium channel; Na_V1, VSGC type 1; CTD, C-terminal domain; LQT3, Long QT syndrome type 3; CaM, calmodulin; HSQC, heteronuclear single quantum spectroscopy; NOESY, nuclear Overhauser effect (NOE) spectroscopy.
↵⁶ K. Yap, University of Toronto.
The atomic coordinates and restraints list (code 2kav) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Complete resonance assignments for Na_V1.2 CTD (BMRB 16032) and backbone resonance assignments for Na_V1.5 CTD (BMRB 16031) have been deposited in the BioMagResBank.
↵^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 HL71165 (to G. S. P.), MSTP 5T32 GM07367 (to V. Z. M.), and R01 GM59273 (to A. G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I and Figs. S1-S3.
↵¹ A Fellow of the Canadian Cystic Fibrosis Foundation. Present address: UCLA-DOE Institute for Genomics and Proteomics, UCLA, Los Angeles, CA 90095-1570.
↵² Established Investigators of the American Heart Association.
↵³ Supported by National Institutes of Health Protein Structure Initiative Grants P50GM62413 and U54GM074958 to the Northeast Structural Genomics Consortium.
- Received September 24, 2008.
- Revision received December 16, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.