title: Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel.
abstract: Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Xinping
authors.initials: X
authors.lastname: Xu
authors.forename: Farzana
authors.initials: F
authors.lastname: Marni
authors.forename: Shengjun
authors.initials: S
authors.lastname: Wu
authors.forename: Zhuocheng
authors.initials: Z
authors.lastname: Su
authors.forename: Faik
authors.initials: F
authors.lastname: Musayev
authors.forename: Sabisha
authors.initials: S
authors.lastname: Shrestha
authors.forename: Changan
authors.initials: C
authors.lastname: Xie
authors.forename: Weihua
authors.initials: W
authors.lastname: Gao
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000737:chemistry / Q000502:physiology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007700:Kinetics; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014981:Xenopus
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D000242:Cyclic AMP