Purchase this article with an account.
X.-Q. Ding, A. V. Matveev, J. B. Fitzgerald, J.-H. Xu, M. P. Malykhina, K. K. Rodgers; The Disease-Causing Mutations in the Carboxyl-Terminus of Cone Cyclic Nucleotide-Gated Channel CNGA3 Subunit Alter the Local Secondary Structure and Interfere With the Channel Active Conformational Change. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1092.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction. Mutations in the cone CNG channel are associated with achromatopsia and progressive cone dystrophy in humans. Over 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them being located in the carboxyl (C)-terminus. This study investigates the defects of two frequently occurring mutations in the C-terminus of CNGA3, the R377W and F488L mutations.
The wild-type (WT) and mutant CNGA3 subunits were expressed in HEK293 cells and the C-termini were expressed and purified from Escherichia coli. Ratiometric measurement of intracellular Ca2+ concentration and electrophysiological recordings were performed to evaluate the channel’s function in response to cGMP; Western blotting using anti-CNGA3 was performed to determine the expression of the WT and mutant channels; immunofluorescence labeling was used to examine the cellular localization of the channel subunits; GST pull-down assay was performed to evaluate the homo and hetero subunit interactions; and circular dichroism spectrum analysis was performed to examine the structural impact of the mutations.
The functional assay showed a concentration-dependent response to cGMP in cells expressing the WT CNGA3 but not in cells expressing the R377W and F488L mutants. We also found an apparent cytosolic aggregation of the mutant channels, compared to the WT. The C-terminus of CNGA3 was shown to be critical for the channel subunit interaction; neither of the mutations had a negative effect on this interaction. Although the R377W and F488L C-termini mutants retained stable, folded structures, the secondary structures of both mutants differed from the WT protein. Furthermore, the WT C-terminus exhibited a significant decrease in α-helical content in response to cGMP, while this allosteric transition was diminished in the two mutants.
This is the first study showing structural impact of the disease-causing mutations in the cone CNG channel subunit. The observed alterations in the local secondary structure and active conformational changes may confer an adverse effect to the channel’s function and cellular processing.
This PDF is available to Subscribers Only