Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel

Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Nav channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Nav channels, exemplified by the Na+-selective channel of bacteria (NaChBac), provides a useful model system for structure–function analysis. Here we report the crystal structure of NavRh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca2+ resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that NavRh is in an ‘inactivated’ conformation. Comparison of NavRh with NavAb reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

[1]  J. Ruppersberg.  Ion Channels in Excitable Membranes , 1996 .

[2]  W. Catterall,et al.  Sequential formation of ion pairs during activation of a sodium channel voltage sensor , 2009, Proceedings of the National Academy of Sciences.

[3]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[4]  B. Wallace,et al.  The pore dimensions of gramicidin A. , 1993, Biophysical journal.

[5]  R. D. Shannon.  Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides , 1976 .

[6]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[7]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[8]  D. Clapham,et al.  The Cation Selectivity Filter of the Bacterial Sodium Channel, NaChBac , 2002, The Journal of general physiology.

[9]  Mark Gerstein,et al.  MolMovDB: analysis and visualization of conformational change and structural flexibility , 2003, Nucleic Acids Res..

[10]  W. Ulbricht,et al.  Sodium channel inactivation: molecular determinants and modulation. , 2005, Physiological reviews.

[11]  M. Cadene,et al.  X-ray structure of a voltage-dependent K 1 channel , 2003 .

[12]  W. Catterall,et al.  The molecular basis of neuronal excitability. , 1984, Science.

[13]  C. Armstrong,et al.  Calcium ion as a cofactor in Na channel gating. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Manuel C. Peitsch,et al.  SWISS-MODEL: an automated protein homology-modeling server , 2003, Nucleic Acids Res..

[15]  B. Hille.  The hydration of sodium ions crossing the nerve membrane. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. MacKinnon,et al.  A gating model for the archeal voltage-dependent K(+) channel KvAP in DPhPC and POPE:POPG decane lipid bilayers. , 2009, Journal of molecular biology.

[17]  D. Clapham,et al.  A Prokaryotic Voltage-Gated Sodium Channel , 2001, Science.

[18]  T Hoshi,et al.  Biophysical and molecular mechanisms of Shaker potassium channel inactivation , 1990, Science.

[19]  D Sodickson,et al.  An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. , 1994, Biophysical journal.

[20]  E. Campbell,et al.  Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment , 2007, Nature.

[21]  Xiao Tao,et al.  A Gating Charge Transfer Center in Voltage Sensors , 2010, Science.

[22]  Francisco Bezanilla,et al.  Voltage-Sensing Residues in the S2 and S4 Segments of the Shaker K+ Channel , 1996, Neuron.

[23]  Torsten Schwede,et al.  BIOINFORMATICS Bioinformatics Advance Access published November 12, 2005 The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling , 2022 .

[24]  H. Fozzard,et al.  Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule. , 1999, Biophysical journal.

[25]  P. Vassilev,et al.  Identification of an intracellular peptide segment involved in sodium channel inactivation. , 1988, Science.

[26]  Samuel H. Wilson,et al.  Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. , 1994, Science.

[27]  Roderick MacKinnon,et al.  Solution structure and phospholipid interactions of the isolated voltage-sensor domain from KvAP. , 2010, Journal of molecular biology.

[28]  E. Campbell,et al.  Voltage Sensor of Kv1.2: Structural Basis of Electromechanical Coupling , 2005, Science.

[29]  M. Harding,et al.  The geometry of metal-ligand interactions relevant to proteins. , 1999, Acta crystallographica. Section D, Biological crystallography.

[30]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[31]  F. Bezanilla,et al.  Currents Related to Movement of the Gating Particles of the Sodium Channels , 1973, Nature.

[32]  Massimo Mantegazza,et al.  Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders , 2010, The Lancet Neurology.

[33]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[34]  A. Brunger.  Version 1.2 of the Crystallography and NMR system , 2007, Nature Protocols.

[35]  G. Tomaselli,et al.  Molecular Motions of the Outer Ring of Charge of the Sodium Channel , 2003, The Journal of general physiology.

[36]  M. Cadene,et al.  X-ray structure of a voltage-dependent K+ channel , 2003, Nature.

[37]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[38]  W. Catterall,et al.  THE CRYSTAL STRUCTURE OF A VOLTAGE-GATED SODIUM CHANNEL , 2011, Nature.

[39]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[40]  Francisco Bezanilla,et al.  Charge Movement Associated with the Opening and Closing of the Activation Gates of the Na Channels , 1974, The Journal of general physiology.

[41]  Roderick MacKinnon,et al.  Contribution of the S4 Segment to Gating Charge in the Shaker K+ Channel , 1996, Neuron.

[42]  M. Gerstein,et al.  The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. , 2000, Nucleic acids research.

[43]  H. Guy,et al.  A putative prokaryote voltage-gated Ca(2+) channel with only one 6TM motif per subunit. , 2001, Biochemical and biophysical research communications.

[44]  R. Aldrich,et al.  Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB , 1990, Science.

[45]  C. Bladen,et al.  The pore, not cytoplasmic domains, underlies inactivation in a prokaryotic sodium channel. , 2005, Biophysical journal.

[46]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[47]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.