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Published on: September 20, 2021
CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179
Nazarul Hasan1, Thomas A Ray1, Ronald G Gregg1
1Department of Biochemistry & Molecular Genetics,University of Louisville,Louisville,Kentucky.
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.
Insights
The CACNA1S antibody, previously thought to mark retinal depolarizing bipolar cells (DBCs), actually cross-reacts with GPR179. This finding questions CACNA1S
Area of Science:
- Neuroscience
- Molecular Biology
- Ophthalmology
Background:
- Voltage-dependent calcium channels, specifically the α1S subunit (Cav1.1) encoded by Cacna1s, are crucial for muscle function.
- CACNA1S was recently reported in retinal depolarizing bipolar cells (DBCs), colocalizing with mGluR6, a key component of DBC signal transduction.
- Previous studies suggested CACNA1S expression in DBCs, with reduced levels in mGluR6 knockout mice.
Purpose of the Study:
- To investigate the functional role of CACNA1S in the DBC signal transduction cascade.
- To validate the expression and localization of CACNA1S in mouse retinas using various genetic models.
- To determine the specificity of antibodies used in previous CACNA1S retinal studies.
Main Methods:
- Examined RNAseq data for novel Cacna1s isoforms in mouse retina.
- Performed immunohistochemistry on wild-type and mutant mouse retinas (Gpr179 nob5, Grm6-/-) using CACNA1S antibodies.
- Conducted Western blot analysis and mass spectrometry on retinal lysates and immunoprecipitated proteins.
- Performed immunohistochemistry and Western blotting on HEK293T cells expressing GPR179.
Main Results:
- Immunohistochemistry showed punctate labeling of CACNA1S at DBC tips in WT retinas, which was absent in Gpr179 nob5 and decreased in Grm6-/- retinas.
- CACNA1S and TRPM1 staining colocalized in WT retinas.
- Western blot and mass spectrometry failed to detect CACNA1S in retinal samples.
- Antibody validation revealed cross-reactivity with GPR179, explaining the observed immunohistochemical staining.
Conclusions:
- The previously reported CACNA1S staining in DBCs is likely due to antibody cross-reactivity with GPR179.
- Western blot and mass spectrometry data do not support the presence of CACNA1S in the DBC signal transduction pathway.
- Caution is advised when interpreting immunohistochemical data for CACNA1S in retinal studies without further validation.
