Abstract
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to many physiological functions, such as rhythmic activity, membrane excitability, and so on. Four subtypes of HCN channels were cloned so far (HCN1-4). We cloned cDNA for HCN4 from the sino-atrial node of the rabbit heart. HCN4 showed the slowest activation kinetics among the subtypes of HCN channels while HCN1 showed the fastest kinetics. It was suggested that this difference in the kinetics may lead to their distinct physiological roles. We showed that S1 transmembrane region and S1-S2 loop are mainly responsible for the difference in the activation kinetics between HCN1 and HCN4 by constructing chimeras, point mutants, and using patch clamp technique. Next, we focused on the structural basis of S1 and replaced each residue in S1 singly with tryptophan (Trp) in HCN1. Five Trp mutants did not show currents in either of mammalian cell or Xenopus oocyte expression system. The periodicity in the effects of the mutations on the expression may demonstrate an alpha-helical structure for S1. The introduction of Trp into the amino acid residues responsible for the different kinetics between HCN1 and 4 also slowed the activation compared with HCN1 wild type. Most of Trp replacements at the conserved amino acid residues among all HCN subtypes abolished currents, which may indicate that these residues face against other transmembrane regions. Unexpectedly, two Trp mutants in the middle of S1 prevented normal channel closure. Taken together, these results show that S1 is crucially involved in the movement of the voltage sensor or in the coupling of the voltage sensor and the activation gate. [Jpn J Physiol 55 Suppl:S45 (2005)]
