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T. I. Rebrik; Ca2+-Dependent Modulation of Cone Photoreceptor cGMP-Gated Channel is Mediated by CNG-Modulin, a Novel Protein. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2040.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the Ca2+ -dependent modulator of the cyclic GMP-gated channel in cone photoreceptors.
Yeast two-hybrid screen of cDNA library prepared from mRNA isolated from striped bass retinas was performed using Matchmaker Two-Hybrid System 3 (Clontech). We used as baits N- and C-terminus cytoplasmic domains of the bass cGMP-gated channels’ β-subunit (CNGB3). Protein-protein interactions were verified using TIRF/FRET assay. Patch-clamp recording, single cell PCR and immunohistochemistry methods were described in detail in our previous work1,2.
Previous physiological studies1,3,4 predicted the existence of a soluble molecule that mediates Ca2+-dependent modulation of the cone cGMP-gated channel; however the molecular identity of this modulator remained unknown. Our yeast two-hybrid search identified a novel protein which binds to the N-terminus of the beta subunit of cone cGMP-gated channel. We called this protein CNG-modulin. Single cell RT-PCR demonstrated that CNG-modulin is expressed in both single and twin cones. Immunohistochemical analysis indicated that CNG-modulin is expressed in fish cones but not rods. TIRF/FRET experiments demonstrated direct interaction between CNG-modulin and CNGB3. Direct functional test of the recombinant CNG-modulin in membrane patches excised from cone outer segments demonstrated that CNG-modulin modulates the sensitivity of the channel to cGMP in the Ca2+-dependent manner. The parameters of the modulation by recombinant CNG-modulin measured in patches were similar to those observed in intact cones containing the endogenous modulator. CNG-modulin shifted the K½ value for channel activation by cGMP from ~90 µM in the absence of Ca2+ to ~330 µM in the presence of saturating Ca2+.
These data suggest that CNG-modulin is the long sought cone channel modulator.1 Rebrik et al., J. Gen. Physiol. 116, 521-534 (2000)2 Pallart et al., Vis. Neurosci. 23, 99-113 (2006)3 Rebrik and Korenbrot, J. Gen. Physiol. 112, 537-48 (1998)4 Rebrik and Korenbrot, J. Gen. Physiol. 123, 63-76 (2004)
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