Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations

J Biol Chem. 2004 Oct 15;279(42):43752-9. doi: 10.1074/jbc.M408747200. Epub 2004 Aug 8.

Abstract

If or Ih, a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229EKGMDSEVY237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K+ channels, complete deletion of the S3-S4 linker (Delta229-237) did not produce functional channels. Similarly, the deletions Delta229-234, Delta232-234, and Delta232-237 also abolished normal current activity. Interestingly, Delta229-231, Delta233-237, Delta234-237, Delta235-237, Delta229-231/Delta233-237, Delta229-231/Delta234-237, and Delta229-231/Delta235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. DeltaV1/2 of Delta229-231, Delta233-237, Delta235-237 > +10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (-35 mV < DeltaV1/2 < -4 mV). Based on these results, we conclude that only Met232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K+ channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene- and cell-based therapies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cyclic Nucleotide-Gated Cation Channels
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • Ion Channels / genetics*
  • Ion Channels / physiology
  • Kinetics
  • Membrane Potentials / physiology
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / physiology
  • Peptide Fragments / chemistry
  • Peptide Fragments / pharmacology*
  • Polymerase Chain Reaction
  • Potassium Channels
  • Protein Conformation
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion

Substances

  • Cyclic Nucleotide-Gated Cation Channels
  • Hcn1 protein, mouse
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • Ion Channels
  • Nerve Tissue Proteins
  • Peptide Fragments
  • Potassium Channels
  • Recombinant Fusion Proteins