MOLECULAR-INTERACTIONS OF 3',5'-CYCLIC PURINE ANALOGS WITH THE BINDING-SITE OF RETINAL ROD ION CHANNELS

Photoreceptor outer segments transduce information about incoming ligh t levels through a class of ion channels that respond directly to chan ges in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C 8 positions was used to examine molecular interactions between the nuc leotide and the channel, The maximal current activated by C2-altered a nalogues in excised membrane patches was less than the current activat ed by cGMP, and the K-0.5, the concentration which activates 50% of th e current in a patch, was increased. Nonpolar C8-substituted cAMP anal ogues activated more current than the parent cAMP with lower K-0.5 val ues. This was in contrast to 8-amino-cAMP, which exhibited greatly red uced activity. The rank order of activity, based on K-0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-ben zylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N-6-Etheno -cAMP and N-6-monobutyryl-cAMP activated a small fraction of the total possible current with high K-0.5 values. Other analogues with alterat ions at N1 or CS positions including N-1-oxide-cAMP, 2-aminopurine rib oside 3',5'-monophosphate, and N-6-monosuccinyl-cAMP do not bind to th e channel, suggesting that interactions with the channel in this regio n are essential for binding. In order to help interpret the changes in maximal current and K-0.5 values compared to cGMP, molecular models o f the active analogues were constructed and then docked into a molecul ar 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 steric ally accommodated within the binding site. No hydrogen bonds were pred icted between the purine rings of cAMP and the pocket; however, Phe 53 3 on the beta 5 strand was predicted to form weak electrostatic intera ctions with C6 substituents on both cAMP and cGMP. The importance of c ontacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6.