title: Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin.
abstract: Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca(2+) ([Ca(2+)](i)). Two regions of Na(V)1.5 have been identified previously as [Ca(2+)](i)-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca(2+) levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca(2+). Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes Na(V)1.5 inactivation, similarly and more extensively than even reducing [Ca(2+)](i). Because the DIII-DIV linker is an essential structure in Na(V)1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe(1520)-Ile(1521)-Phe(1522)) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: Na(V)1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na(+) channel function in genetically altered channels and under pathophysiologic conditions where [Ca(2+)](i) impacts cardiac conduction.
authors.affiliation: Departments of Anesthesiology, Pharmacology, Medicine, Biochemistry, and Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, USA. franck.potet@vanderbilt.edu
authors.forename: Franck
authors.initials: F
authors.lastname: Potet
authors.forename: Benjamin
authors.initials: B
authors.lastname: Chagot
authors.forename: Mircea
authors.initials: M
authors.lastname: Anghelescu
authors.forename: Prakash C
authors.initials: PC
authors.lastname: Viswanathan
authors.forename: Svetlana Z
authors.initials: SZ
authors.lastname: Stepanovic
authors.forename: Sabina
authors.initials: S
authors.lastname: Kupershmidt
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Balser
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D053840:Brugada Syndrome / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D062554:NAV1.5 Voltage-Gated Sodium Channel; D019906:Nuclear Magnetic Resonance, Biomolecular; D055550:Protein Stability; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D015222:Sodium Channels; D002118:Calcium