Tryptophan-scanning mutagenesis in the S1 domain of mammalian HCN channel reveals residues critical for voltage-gated activation.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential regulators in rhythmic activity, membrane excitability and synaptic transmission. There are four subtypes in mammals (HCN1-4); HCN4 has the slowest activation kinetics and HCN1 the fastest. Although voltage gating originates with the voltage-dependent motion of the S4 segment, the different activation kinetics between HCN1 and HCN4 are generated mainly by S1 and the S1-S2 loop. In this study, we investigate the structural basis of the ability of S1 to affect activation kinetics by replacing each individual S1 residue in HCN1 with a tryptophan (Trp) residue, a Trp perturbation scan. Robust currents were generated in 11 out of 19 Trp mutants. Hyperpolarization-activated currents were not detected in four mutants, and two other mutants generated only small currents. Presence or absence of current reflected the predicted alpha-helical structure of the S1 transmembrane segment. Tryptophan replacements of residues responsible for the different kinetics between HCN1 and HCN4 made the activation kinetics slower than the wild-type HCN1. Tryptophan mutations introduced in the middle of S1 (L139W and V143W) prevented normal channel closure. Furthermore, a negatively charged residue at position 139 (L139D) induced a positive voltage shift of activation by 125 mV. Thus, L139 and V143 probably face a mobile part of the S4 voltage sensor and may interact with it. These results suggest that the secondary structure of S1 is alpha-helical and profoundly affects the motion of the voltage sensor.
Ishii
Takahiro M
TM
Department of Physiology, Kyoto University, Kyoto 606-8501, Japan.
Nakashima
Noriyuki
N
Ohmori
Harunori
H
D000595:Amino Acid Sequence; D000818:Animals; D054815:Cyclic Nucleotide-Gated Cation Channels; D004594:Electrophysiology; D005260:Female; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics*; D009865:Oocytes; D015221:Potassium Channels; D017434:Protein Structure, Tertiary / Q000235:genetics*; D014364:Tryptophan; D014982:Xenopus laevis
D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D014364:Tryptophan