title: CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179.
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.
authors.affiliation: Department of Biochemistry & Molecular Genetics,University of Louisville,Louisville,Kentucky.
authors.forename: Nazarul
authors.initials: N
authors.lastname: Hasan
authors.affiliation: Department of Biochemistry & Molecular Genetics,University of Louisville,Louisville,Kentucky.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Ray
authors.affiliation: Department of Biochemistry & Molecular Genetics,University of Louisville,Louisville,Kentucky.
authors.forename: Ronald G
authors.identifier: 0000-0001-5805-224X
authors.initials: RG
authors.lastname: Gregg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D020746:Calcium Channels, L-Type / Q000235:genetics* / Q000276:immunology*; D003429:Cross Reactions; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007153:Immunologic Deficiency Syndromes; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D000081207:Primary Immunodeficiency Diseases; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D043562:Receptors, G-Protein-Coupled / Q000276:immunology*; D012160:Retina / Q000378:metabolism*; D051245:Retinal Bipolar Cells / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C529218:CACNA1S protein, mouse; D020746:Calcium Channels, L-Type; C575060:GPR179 protein, mouse; D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled
keywords: Antibody cross-reactivity; Bipolar cells; CACNA1S; GPR179; Retina