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Tryptophan‐scanning mutagenesis in the S1 domain of mammalian HCN channel reveals residues critical for voltage‐gated activation
Author(s) -
Ishii Takahiro M.,
Nakashima Noriyuki,
Ohmori Harunori
Publication year - 2007
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2006.124297
Subject(s) - tryptophan , chemistry , kinetics , biophysics , hyperpolarization (physics) , gating , transmembrane domain , pas domain , membrane potential , mutant , ion channel , biochemistry , stereochemistry , biology , membrane , amino acid , nuclear magnetic resonance spectroscopy , physics , quantum mechanics , gene , transcription factor , receptor
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 α‐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 α‐helical and profoundly affects the motion of the voltage sensor.