Verlag des Forschungszentrums Jülich

JUEL-3815
Vantler, Marius
Proteinkinase C-abhängige Phosphorylierung zyklisch-Nukleotid-gesteuerter Ionenkanäle aus den Sehzellen des Rindes
107 S., 2000



In retinal rod and cone photoreceptor cells cyclic nucleotide-gated (CNG) channels mediate the electrical response to stimulation by light. CNG channels are composed of different subunits, designated CNC[alpha] or CNC[beta] subunits. CNG channels are gated by the internal messenger molecule cGMP and are modulated by small Ca²⁺-binding proteins. This PhD- thesis shows that the CNG channels from rod and cone photoreceptor cells are phosphorylated by protein kinase C (PKC). PKC-dependent phosphorylation of homomeric heterologously expressed cone CNG channels composed of CNC[alpha]2 subunits was investigated electrophysiologically in inside-out patches. Phosphorylation of two serine residues (serine 577 and serine 579) in the cGMP-binding pocket of the CNC[alpha]2 subunit decreases the ligand sensitivity of cone CNG channels by a factor of 2 -3. The decrease in ligand sensitivity is completely abolished in mutant channels in which the two serine residues were replaced by alanine.

The auxiliary CNC[beta]la subunit of the rod CNG channel is phosphorylated by PKC too. The in vitro phosphorylation of different recombinant fusion proteins shows that the phosphorylation sites are localized in the vicinity of the N-terminal calmodulin (CaM)-binding domain. Binding of CaM to the CNC[beta]la subunit reduces the degree of phosphorylation, whereas phosphorylation did not prevent CaM from binding.

GARP2 is an N-terminal splice form of the CNC[beta]la subunit. In contrast to the CNC[alpha]2 and CNC[beta]la subunit, GARP2 is not phosphorylated by PKC. Gel filtration experiments showed that GARP2 binds the light-activated cGMP-hydrolyzing enzyme phosphodiesterase. In this way GARP2 may organize an "adaptational" signa1ing complex at the disk rim of the rod photoreceptor cell that controls cGMP turnover during daylight.

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