June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
CACNA1S expression, structure and function in retinal depolarizing bipolar cells
Author Affiliations & Notes
  • Thomas Ray
    Biochemistry & Molecular Biology, University of Louisville, Louisville, KY
  • Nazarul Hasan
    Biochemistry & Molecular Biology, University of Louisville, Louisville, KY
  • Maureen McCall
    Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY
  • Ronald Gregg
    Biochemistry & Molecular Biology, University of Louisville, Louisville, KY
  • Footnotes
    Commercial Relationships Thomas Ray, None; Nazarul Hasan, None; Maureen McCall, None; Ronald Gregg, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 6159. doi:https://doi.org/
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      Thomas Ray, Nazarul Hasan, Maureen McCall, Ronald Gregg; CACNA1S expression, structure and function in retinal depolarizing bipolar cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6159. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To identify and characterize the function of new components of the GRM6 signal cascade in depolarizing bipolar cells (DBCs). CACNA1S, the alpha subunit of voltage-dependent calcium channels (VDCCs), is expressed at the tips of the DBCs and colocalizes with GRM6. CACNA1S is the pore forming subunit of the VDCCs in skeletal muscle, where it couples to the Ryanodine receptor to regulate calcium entry into the cell. CACNA1S protein expression appears absent or downregulated at the tips of the DBCs in two models of complete Congenital Stationary Nightblindness (cCSNB), implicating a possible role in the GRM6 signal cascade.

Methods: Immunohistochemistry was performed to visualize protein expression of CACNA1S in the DBCs of wildtype and Gpr179nob5/nob5 retinas. Analysis of next generation RNASeq data was used to verify Cacna1s gene expression in the retina. Cloning of cDNA representing Cacna1s from the retina was performed to determine the predicted amino acid sequence. Gene expression of Cacna1s was analyzed by qRT-PCR and compared to expression of other genes in the GRM6 signaling cascade (Grm6, Gpr179 and Nyx). The level of various alternative transcripts was determined. Protein expression was analyzed by western blot analysis.

Results: CACNA1S protein is absent from the DBCs in Gpr179nob5/nob5 mouse retinas as detected by immunohistochemistry. Conversely, in Grm6-/- retinas, CACNA1S protein is localized correctly, although the level is decreased. Other proteins absent from the DBC tips in the Gpr179nob5/nob5 retina include RGS7, RGS11 and Gβ5, all of which are involved in the bipolar cell light response. RNA Seq data from mouse retinas identified a novel isoform of Cacna1s. The novel isoform was cloned from retina cDNA and expressed in COS-7 cells.

Conclusions: CACNA1S protein is expressed at the tips of the DBCs and its localization is dependent on the expression of GPR179. The retina isoform of Cacna1s is novel and is significantly different from the gene expressed in skeletal muscle. Elimination or downregulation of CACNA1S from the tips of the DBCs in various mouse models of cCSNB indicate it is a critical component of the GRM6 signaling cascade in DBCs.

Keywords: 435 bipolar cells • 554 immunohistochemistry • 533 gene/expression  
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