Involvement of Separate Regions of the NAV Channel DIII-IV Linker Revealed through State Dependent Trapping with Genetically Encoded Photochemistry

Abstract

Fast inactivation in voltage-gated sodium channels results from rapid and reversible conformational changes in the cytoplasmic linker between domains III and IV (DIII-IV) and their putative receptor in the pore region of the channel. Introduced or acquired mutations in this complex lead to defective channel inactivation, yet the precise structural basis for these interactions has not yet been fully described. Using the photocrosslinking (PC) unnatural amino acid p-benzoyl-L-phenyl alanine (pBpa) it is possible to trap close structural interactions (∼3 A) during transient conformational rearrangements thus mapping structural reorganisations associated with different states. The rates at which crosslinking reactions occur can report on the affinity of such interactions. We have incorporated the pBpa in mammalian cells at the putative inactivation peptide (IFMT) motif of the DIII-IV linker and demonstrated state dependent trapping of the transient conformations associated with fast inactivation, with different phenotypes and rates of trapping revealing distinct environmental changes at each site. Incorporation of pBpa at two tyrosine residues, Y1494 and Y1495 of NaV1.5, which have previously been implicated in the coupling of activation and inactivation and to interact with Calmodulin produced channels with normal gating and robust expression. Interestingly, the observed state dependent trapping upon irradiation with UV light during patch clamp experiments was functionally distinct from the state-dependent cross-linking interactions observed within the IFM locus. Thus, trapping at these sites suggests a more extensive involvement of the DIII-DIV linker in fast inactivation gating beyond the canonical IFMT motif.