Molecular Interactions of 3',5'-Cyclic Purine Analogs with the Binding Site of Retinal Rod Ion Channels

Abstract

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.

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

“…1) Substitutions at the C-8 position of the purine ring increase bulkiness and shift the conformational equilibrium toward the syn-form (19,39,47,48). If cGMP bound to the noncatalytic sites in the syn-conformation, it would be expected that 8-substituted analogs might show increased binding affinity compared with cGMP, as has been observed for the binding sites of the cyclic nucleotide-gated ion channel (16,18,19) and the cGMP-dependent protein kinase (15). In both of these proteins, cGMP is believed to occupy the binding site in the syn-conformation (18,49,50).…”

“…If cGMP bound to the noncatalytic sites in the syn-conformation, it would be expected that 8-substituted analogs might show increased binding affinity compared with cGMP, as has been observed for the binding sites of the cyclic nucleotide-gated ion channel (16,18,19) and the cGMP-dependent protein kinase (15). In both of these proteins, cGMP is believed to occupy the binding site in the syn-conformation (18,49,50). Instead, almost all substitutions at the C-8 position substantially reduce the binding affinity of cGMP analogs to the PDE noncatalytic sites (Table III), consistent with an anti-conformation of cGMP in the binding pocket.…”

“…For the case of three comparisons of short versus longer chain length substituents at the C-2 position (24 versus 15, 17 versus 11, and 20 versus 16), the longer alkyl group demonstrated a statistically significant higher binding affinity than the shorter alkyl chain. The moderate binding of N 2 -phenyl-cGMP (12), N 2 -biphenyl-cGMP (13), and N 2 -benzyl-cGMP (18) demonstrates that overall bulkiness is not the limiting factor for tight binding at the C-2 position; these three compounds are bulky but still retain their capacity for H bonding. The fact that methyl, ethyl, phenyl, biphenyl, and hexyl substituents at position 2 all bound with moderate affinity indicates that this region of the binding pocket is not spatially constrained, at least in one of the directions occupied by the two N 2 hydrogen atoms in the cGMP molecule.…”

Mutating Three Residues in the Bovine Rod Cyclic Nucleotide-Activated Channel Can Switch a Nucleotide from Inactive to Active

“…Previous studies using nucleotide analogs have shown that the binding domain is remarkably tolerant of changes in the purine ring substituents. Analogs with a thio substituent as 6-thio-cGMP, a monobutyryl as N 6 -monobutyryl cAMP, or rings as 1-N 6 -etheno cAMP (Scott and Tanaka, 1995;Tanaka et al, 1989) and PET-cGMP (Wei et al, 1996), all activate currents in rod channels. Due to this tolerance for changes in both size and charge at various positions on the purine, the most interesting analogs are those that fail to activate CNG channels.…”

“…Notably, the only inactive purine-modified analogs identified to date are modified at the C 6 or N 1 positions. Two of these analogs have single atom changes: 2-aminopurine riboside 3Ј-5Ј-monophosphate (2-amino-cPNP), which has a hydrogen in place of the oxygen of cGMP at C 6 (Tanaka et al, 1989), and N 1 -oxide cAMP with an oxygen instead of hydrogen at the N 1 position (Scott and Tanaka, 1995). Neither analog binds to rod channels, as shown by cGMP competition studies.…”