Calmodulin modulation of the cGMP-gated channel of photoreceptor rod cell

During the past decade, great advances have been made towards deciphering the molecular mechanism of vision. Findings from various laboratories have led to the establishment of the visual cascade and the identification of the main members of this pathway. However, little is known so far about the intricate regulation of the various enzymes involved, in particular, the roles of Ca₂ and Ca₂-binding proteins. This thesis describes the finding of a novel regulation of the visual transduction process through the Ca²⁺-calmodulin dependent modulation of the cGIVIP-gated channels. An unknown 17/20 kDa protein found in the EDTA extract of bovine rod outer segments (ROS) was purified and identified as calmodulin by a variety of biochemical and immunochemical studies. The cGMP-gated channel complex, which plays an important role in phototransduction, was found to be the major calmodulin binding protein in ROS membranes by calmodulin affinity chromatography and Western blotting analysis with iodinated calmodulin. This association between calmodulin and the channel complex, which is mediated through a high molecular weight channel associated protein, was found to be conserved among various species. The effect of calmodulin on the ROS cGIVIP-gated channel activity was investigated by a Ca²⁺ influx assay using ROS membrane vesicles pre-loaded with Arsenazo III dye. In the absence of calmodulin, the channel displayed an apparent Km of 19 ± 0.4 µM for cGMP, while in the presence of calmodulin, its apparent Km increased to 33 ± 2 µM. Similar shifts in the Km of the channel for cO1VIP were observed using extruded ROS membrane vesicles containing either dichiorophosphonazo III or neutral red. This effect is specific for calmodulin since otherCa²⁺ binding proteins such as bovine recoverin and brain S-100 caused no change on channel activity. The calmodulin mediated shift in the affinity of the channel for cGMP could be reversed by mastoparan, a peptide inhibitor of calmodulin. In addition to Ca²⁺, calmodulin also appeared to affect the translocation of various monovalent and divalent cations across the ROS membrane. Calmodulin modulation of the channel was observed to occur within a physiological Ca²⁺ range of 20-300 nM. The calmodulin effect was shown to be mediated through the 240 kDa channel associated protein as determined by a Ca²⁺ efflux assay using the immunoaffinity purified channel complex reconstituted into lipid vesicles. In the absence of calmodulin, the reconstituted channel complex displayed a Km of 33 µM for cGMP, while in the presence of calmodulin, the Km increased to 44 µM. N-terminal sequence analysis of a 105 kDa calmodulin-binding fragment of the 240 kDa protein revealed a sequence that matched with the recently cloned β-subunit of the human rod cGMP-gated channel. In summary, the presence of calmodulin in ROS has been detected. This protein binds preferentially to the 13-subunit of the cGMP-gated channel complex and can modulate the affinity of the channel for cGMP. A model of the calmodulin binding to the channel complex is presented and the importance of the calmodulin mediated regulation of the channel activity is discussed.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat