CAGED COMPOUNDS OF HYDROLYSIS-RESISTANT ANALOGS OF CAMP AND CGMP - SYNTHESIS AND APPLICATION TO CYCLIC NUCLEOTIDE-GATED CHANNELS

Photolabile compounds which rapidly release cAMP or cGMP after photoly sis are widely used for in situ studies of signaling pathways inside c ells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrob enzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br -cGMP (caged 8-Br-cCMP), which respectively release the hydrolysis-res istant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiol ogical studies was examined in a mammalian cell line expressing the cy clic nucleotide-gated (CNG) ion channel of bovine olfactory sensory ne urons. The synthesis procedure resulted in diastereomeric mixtures whi ch were chromatographically separated into the axial and equatorial is omers of caged 8-Br-cAMP and of caged 8-Br-cGMP. The axial isomers whi ch have a higher solubility and better solvolytic stability than the e quatorial forms were used for experiments with CNG channels. Flashes o f UV light produced steps in the concentration of 8-Br-cGMP which acti vated currents through CNG channels. Concentration steps inside the ce ll could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtain ed with caged 8-Br-cAMP. Control experiments with caged cGMP showed th at flash-induced currents decayed within a few minutes because photore leased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consisten t with a bimolecular reaction between channel and ligand.