Functional characterization of a metal ion coordination site in HCN4.

Abstract

Hyperpolarization-activated cyclic nucleotide sensitive (HCN4) channels are the molecular correlate of the funny current (If), which is required for cardiac pacemaking. HCN4 channels are potentiated by cAMP, which shifts the voltage-dependence of activation to more positive potentials by binding directly to a cytoplasmic cyclic nucleotide binding domain (CNBD). The CNBD is connected to the transmembrane domains of the channel via the C-linker. Recent cryo-EM structures of HCN4 in the presence and absence of cAMP identified an isoform-specific metal ion coordination site between the C-linker and the S4-S5 linker in HCN4. The metal coordination site consists of four residues, two in the S4-S5 linker and two in the C-linker. These residues are conserved across mammalian HCN channel isoforms, yet divalent cations such as Mg2+ only regulate HCN4. Here, we examined how mutations in the metal coordination site and non-conserved residues in the C-linker affect the ability of divalent cations to regulate HCN4. In preliminary experiments, we found that mutation of two histidines in the coordination site eliminated the ability of Mg2+ but not Ca2+ to regulate HCN4. Ongoing experiments use a channel in which all four of the coordinating residues are changed to alanine to test the hypothesis that Ca2+ acts at the same site, but is coordinated differently than Mg2+. We are also examining divalent regulation of mutant channels in which two non-conserved residues in the C-linker of HCN4 are mutated to the homologous residues in HCN2. Preliminary data suggest that the unique C-linker in HCN4 may be required for regulation by divalent cations, possibly by determining the orientation of the metal ion coordination site.