Molecular interactions of 3',5'-cyclic purine analogues with the binding site of retinal rod ion channels.
Photoreceptor outer segments transduce information about incoming light levels through a class of ion channels that respond directly to changes in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C8 positions was used to examine molecular interactions between the nucleotide and the channel. The maximal current activated by C2-altered analogues in excised membrane patches was less than the current activated by cGMP, and the K0.5, the concentration which activates 50% of the current in a patch, was increased. Nonpolar C8-substituted cAMP analogues activated more current than the parent cAMP with lower K0.5 values. This was in contrast to 8-amino-cAMP, which exhibited greatly reduced activity. The rank order of activity, based on K0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-benzylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N6-Etheno-cAMP and N6-monobutyryl-cAMP activated a small fraction of the total possible current with high K0.5 values. Other analogues with alterations at N1 or C6 positions including N1-oxide-cAMP, 2-aminopurine riboside 3',5'-monophosphate, and N6-monosuccinyl-cAMP do not bind to the channel, suggesting that interactions with the channel in this region are essential for binding. In order to help interpret the changes in maximal current and K0.5 values compared to cGMP, molecular models of the active analogues were constructed and then docked into a molecular model of the cyclic nucleotide binding site of the retinal channel. This model, proposed by Kumar and Weber [(1992) Biochemistry 31, 4643-4649], was based on the crystal structure of cAMP bound to catabolite activator protein. Our modeling showed that the analogues were sterically accommodated within the binding site. No hydrogen bonds were predicted between the purine rings of cAMP and the pocket; however, Phe 533 on the beta 5 strand was predicted to form weak electrostatic interactions with C6 substituents on both cAMP and cGMP. The importance of contacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6.
Scott
S P
SP
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Tanaka
J C
JC
D000557:Ambystoma; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D012374:Rod Cell Outer Segment / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D008024:Ligands; D006152:Cyclic GMP