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S. Michalakis, X. Zong, E. Becirovic, M. Biel; Functional Characterization of a Retinitis Pigmentosa-Associated CNGB1 Mutation (G993V). Invest. Ophthalmol. Vis. Sci. 2008;49(13):2997. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The rod cyclic nucleotide-gated (CNG) channel is a heteromer consisting of three CNGA1 and one CNGB1a subunit. In this study, we characterized the functional consequences of a point mutation in the CNGB1 gene (G to T at position nt2978 of human CNGB1), that leads to a glycine-valine replacement in the cyclic nucleotide binding domain (G993V) of the B1a protein. This point mutation was previously described in a French family with severe recessive Retinits pigmentosa (RP).
HEK293 cells where cotransfected with CNGA1 (A1) and either wild type (B1a) or mutant CNGB1a (B1aG993V). The channels were analyzed by combining site-directed mutagenesis, electrophysiology, biochemistry and immunocytochemistry.
Transfected HEK293 cells expressed the B1aG993V protein. There was also evidence for trafficking of B1aG993V to the plasma membrane. Currents recorded after transfection with A1/B1aG993V did not differ from A1/B1a currents with repect to their apparent cGMP affinity. Biochemical assays indicated that A1 and B1aG993V formed a stable protein complex. Surprisingly, however, the functional properties of A1/B1aG993V channels were virtually identical to those of homomeric A1 channels. By contrast, channel features typically confered by B1a, such as sensitivity to L-cis-diltiazem, single-channel flicker or sensitivity to Ca2+-calmodulin, were absent in A1/B1aG993V. Together, these data suggest that A1/B1a993V channels, while formed, are functionally inactive. We propose that currents observed after coexpression of A1 and B1aG993V solely derive from homomeric A1 channels that are formed along with heteromeric channels. We present preliminary data suggesting that B1aG993V can be functionally rescued by truncating its long cytosolic N-terminus.
Our results indicate that the G993V mutation strongly affects the CNG channel properties. We propose that assembling of B1aG993V with A1 leads to functionally inactive channels. Thus, in a physiological setting, the G993V mutation is likely to be equivalent to the total loss of CNGB1 function explaining the severe phenotype of patients suffering from this mutation.
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