Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site.
cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.
Wei
J Y
JY
Department of Ophthalmology, Yale University School of Medicine, New Haven, Connecticut 06520-8061, USA.
Cohen
E D
ED
Yan
Y Y
YY
Genieser
H G
HG
Barnstable
C J
CJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry; D015394:Molecular Structure; D009865:Oocytes / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D015335:Molecular Probes; D011994:Recombinant Proteins; D013873:Thionucleotides; C098740:8-bromo-beta-phenyl-1,N(2)-ethenoguanosine 3',5'-cyclic monophosphorothioate; D006152:Cyclic GMP