Determinants of activation kinetics in mammalian hyperpolarization-activated cation channels.
1. The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS-7). 2. The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at -100 mV. 3. In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1-S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics. 4. The S1 domain and S1-S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1-S2 region of HCN1 by that of HCN4 resulted in a 10- to 20-fold slowing. 5. The results indicate that S1 and S1-S2, and S6-CNBD are the crucial components for the activation gating of HCN channels.
Ishii
T M
TM
Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Takano
M
M
Ohmori
H
H
D000818:Animals; D019556:COS Cells; D002412:Cations / Q000378:metabolism*; D002678:Chimera; D054815:Cyclic Nucleotide-Gated Cation Channels; D004594:Electrophysiology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D017354:Point Mutation; D015221:Potassium Channels; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D013997:Time Factors
D002412:Cations; D054815:Cyclic Nucleotide-Gated Cation Channels; C578665:HCN1 protein, human; C119587:HCN4 protein, human; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels