Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations.
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.
Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USa.
Suk Ying
SY
Tsang
Heinte
H
Lesso
Ronald A
RA
Li
D000595:Amino Acid Sequence; D000818:Animals; D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D015221:Potassium Channels; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins