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EC Steele, X Chen, PR MacLeish; Ca2+ Channel Alpha1C Subunit Specifically Expressed in Bipolar Cells of Retina in Higher Vertebrates . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3771.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To compare the molecular expression pattern of the α1C subunit of voltage-gated, L-type Ca2+ channels within the retina of various vertebrates. Methods:Frozen sections, dissociated cells, membrane proteins, and total RNA were prepared from salamander, rat, and monkey retinae. Sections and cells were immunostained with primary antibodies against α1C, PKC-α, and SV2. Fluorescent secondary antibodies and confocal microscopy were used to image immunoreactivity. Western analysis of membrane proteins was performed using these antibodies and standard protocols. Isoform-specific primers were used in RT-PCR reactions on total retinal RNA from rat and monkey to amplify α1C mRNA. Endonuclease restriction and subsequent cloning and sequencing of these amplimers were used to confirm the molecular identity. Results:In the salamander, α1C immunoreactivity appeared highest in the outer and inner plexiform layers and showed a high degree of overlap with SV2 immunoreactivity on photoreceptor and bipolar terminals. These results are consistent with previous reports. However, in rat and monkey retina, α1C immunoreactivity appeared restricted to rod-bipolar cells, showing extensive colocalization with the on-bipolar cell marker PKC-α. Western blot analysis confirmed specific immunoreactivity of the antibodies, but the molecular weight of the identified proteins was significantly smaller than reported for α1C in other tissues. RT-PCR and subsequent sequencing confirmed the presence of high levels of α1C mRNA within both the rat and monkey retinae. Conclusion:These results demonstrate evolutionary diversity in the expression of the α1C Ca2+ channel subunit in the retina of distinct vertebrates. Important functional differences may underlie this species variation. The significant difference in the molecular weight of the vertebrate retinal α1C also indicates that it could be a retina-specific splice variant, distinct from the larger forms found in brain. The use of such splice variants would allow for even greater functional diversification of Ca2+ channel subunits. Mutations of the recently identified α1F Ca2+ channel subunit, which is highly expressed in vertebrate photoreceptors, have been linked to congenital night blindness in humans. We propose that similar mutations in α1C might underlie forms of human blindness associated with bipolar cell dysfunction.
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