| Summary: | 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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