Distinct structural determinants of efficacy and sensitivity in the ligand-binding domain of cyclic nucleotide-gated channels.
Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a beta-roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oo-cytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-beta 8" region (the C-terminal end of the beta-roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the beta-roll and the C-helix at different points in the mechanism.
Center for Neurobiology & Behavior, Columbia University, New York, New York 10032, USA. ecy4@columbia.edu
Edgar C
EC
Young
Natalia
N
Krougliak
D000595:Amino Acid Sequence; D000818:Animals; D002397:Catfishes; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D008024:Ligands; D000242:Cyclic AMP; D006152:Cyclic GMP