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Insights on the regulation of the cgmp-gated channel by ca++/calmodulin and on the phosphorylation of the b-subunit by a ckII-like protein kinase Warren, Rene


In vertebrate rod photoreceptors, the cGMP-gated channel plays a crucial role by transducing light-induced changes in cGMP into electrical impulses. Regulation of rod cGMPgated channels by phosphorylation and Ca²⁺/calmodulin has been examined here. The activity of a protein kinase responsible for the phosphorylation of the P- but not the a-subunit was found in the cytosol fraction of purified rod outer segment (ROS) preparations. Phosphorylation occurs mainly on the C-terminal portion of the P-subunit and only on serine residues. cGMP-gated channels purified from ROS, phosphorylated by endogenous ROS kinase(s) and reconstituted into phospholipid vesicles did not exhibit a differential cGMP sensitivity behavior when compared to an unphosphorylated control. In addition, no common protein kinase regulators, second messengers or physiological conditions tested altered the phosphate incorporation into the β-subunit. Taken together with inhibitor mapping experiments and phosphorylation assays using GTP as phosphate donor, these results revealed that the endogenous ROS protein kinase responsible for the P-subunit phosphorylation exhibits properties in common with casein kinase II (CK2). Immunofluorescence microscopy of bovine and rat retina sections labeled with either monoclonal or polyclonal anti-CK2 antibodies have confirmed the presence of a CK2-like protein kinase in ROS. In SDS-polyacrylamide gels, the ROS CK2 migrates slightly slower than its human recombinant homolog and may therefore represent a novel CK2 isoform or a CK2a variant of the enzyme. Further phosphorylation studies using the human recombinant CK2 demonstrates that the rod cGMPgated channel is effectively a target for CK2 in vitro. The involvement of ROS calmodulin (CaM) in phototransduction was also studied. ROS calmodulin was found associated with the native channel complex under similar calcium conditions to those occurring in dark-adapted ROS. Furthermore, calmodulin did not co-purify with the native channel when ROS membranes were washed in the absence of calcium but in the presence of EDTA. cGMP-dependent Ca²⁺-efflux assays and CaM-Sepharose chromatography also confirmed that CaM is the authentic ligand for these CaM-binding sites, since no other calcium binding proteins beside calmodulin have been detected in ROS using these techniques. These observations support a model in which calmodulin is a physiological modulator of the cGMP-gated channel and this regulation may be important for the adaptation and recovery of the photoreceptors by facilitating the reopening of the channels.

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