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CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179

Published online by Cambridge University Press:  31 May 2016

NAZARUL HASAN ,
THOMAS A. RAY  and
RONALD G. GREGG Open the ORCID record for RONALD G. GREGG [Opens in a new window]
NAZARUL HASAN
Affiliation:
Department of Biochemistry & Molecular Genetics, University of Louisville, Louisville, Kentucky
THOMAS A. RAY
Affiliation:
Department of Biochemistry & Molecular Genetics, University of Louisville, Louisville, Kentucky
RONALD G. GREGG*
Affiliation:
Department of Biochemistry & Molecular Genetics, University of Louisville, Louisville, Kentucky Department of Ophthalmology & Visual Sciences, University of Louisville, Louisville, Kentucky
*
*Address correspondence to: Ronald G. Gregg, Department of Biochemistry & Molecular Genetics, University of Louisville, 319 Abraham Flexner Way, Louisville, KY 40202. Email: [email protected]
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Abstract

Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 −/− mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.

Keywords

CACNA1SGPR179RetinaAntibody cross-reactivityBipolar cells
Type
Brief Communication
Information
Visual Neuroscience , Volume 33 , 2016 , E009
Copyright
Copyright © Cambridge University Press 2016 

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