Helical Secondary Structure of the External S3-S4 Linker of Pacemaker (HCN) Channels Revealed by Site-dependent Perturbations of Activation Phenotype

• Published in: The Journal of biological chemistry 2003-06, Vol.278 (25), p.22290-22297
• Main Authors: Lesso, Heinte, Li, Ronald A
• Format: Article
• Language: English
• Subjects: Algorithms; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Cloning, Molecular; Cyclic Nucleotide-Gated Cation Channels; HCN1 protein; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Ion Channels - chemistry; Ion Channels - physiology; Membrane Potentials - physiology; Mice; Models, Molecular; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Patch-Clamp Techniques; Polymerase Chain Reaction; Potassium Channels; Protein Structure, Secondary; Recombinant Proteins - chemistry; Sequence Deletion
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302466200

I f , encoded by the h yperpolarization-activated c yclic n ucleotide-modulated channel family (HCN1–4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229–237) and studying their effects. We found that the mutations K230A (–62.2 ± 3.4 mV versus –72.2 ± 1.7 mV of wild type (WT)), G231A (–64.4 ± 1.3 mV), M232A ( V ½ = –63.1 ± 1.1 mV), and E235G (–65.4 ± 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (1 kcal/mol) clustered on one side.