Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-08T15:30:01.708Z Has data issue: false hasContentIssue false

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

Published online by Cambridge University Press:  31 May 2016

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]

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.

Type
Brief Communication
Copyright
Copyright © Cambridge University Press 2016 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Audo, I., Bujakowska, K., Orhan, E., Poloschek, C.M., Defoort-Dhellemmes, S., Drumare, I., Kohl, S., Luu, T.D., Lecompte, O., Zrenner, E., Lancelot, M.E., Antonio, A., Germain, A., Michiels, C., Audier, C., Letexier, M., Saraiva, J.P., Leroy, B.P., Munier, F.L., Mohand-Said, S., Lorenz, B., Friedburg, C., Preising, M., Kellner, U., Renner, A.B., Moskova-Doumanova, V., Berger, W., Wissinger, B., Hamel, C.P., Schorderet, D.F., De Baere, E., Sharon, D., Banin, E., Jacobson, S.G., Bonneau, D., Zanlonghi, X., Le Meur, G., Casteels, I., Koenekoop, R., Long, V.W., Meire, F., Prescott, K., de Ravel, T., Simmons, I., Nguyen, H., Dollfus, H., Poch, O., Leveillard, T., Nguyen-Ba-Charvet, K., Sahel, J.A., Bhattacharya, S.S. & Zeitz, C. (2012). Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness. American Journal of Human Genetics 90, 321330.Google Scholar
Bech-Hansen, N.T., Naylor, M.J., Maybaum, T.A., Sparkes, R.L., Koop, B., Birch, D.G., Bergen, A.A., Prinsen, C.F., Polomeno, R.C., Gal, A., Drack, A.V., Musarella, M.A., Jacobson, S.G., Young, R.S. & Weleber, R.G. (2000). Mutations in NYX, encoding the leucine-rich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness. Nature Genetics 26, 319323.Google Scholar
Brooks, M.J., Rajasimha, H.K., Roger, J.E. & Swaroop, A. (2011). Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl(−/−) retinal transcriptomes. Molecular Vision 17, 30343054.Google ScholarPubMed
Chaudhari, N. (1992). A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice. Journal of Biological Chemistry 267, 2563625639.Google Scholar
Dryja, T.P., McGee, T.L., Berson, E.L., Fishman, G.A., Sandberg, M.A., Alexander, K.R., Derlacki, D.J. & Rajagopalan, A.S. (2005). Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6. Proceedings of the National Academy of Sciences of the United States of America 102, 48844889.CrossRefGoogle ScholarPubMed
Gregg, R., Lukasiewicz, P., Peachey, N., Sagdullaev, B. & McCall, M. (2003). Nyctalopin is required for signaling through depolarizing bipolar cells in the murine retina. Investigative Ophtalmology and Visual Science 44, 4180.Google Scholar
Gregg, R.G., Kamermans, M., Klooster, J., Lukasiewicz, P.D., Peachey, N.S., Vessey, K.A. & McCall, M.A. (2007). Nyctalopin expression in retinal bipolar cells restores visual function in a mouse model of complete X-linked congenital stationary night blindness. Journal of Neurophysiology 98, 30233033.Google Scholar
Koike, C., Obara, T., Uriu, Y., Numata, T., Sanuki, R., Miyata, K., Koyasu, T., Ueno, S., Funabiki, K., Tani, A., Ueda, H., Kondo, M., Mori, Y., Tachibana, M. & Furukawa, T. (2010). TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade. Proceedings of the National Academy of Sciences of the United States of America 107, 332337.CrossRefGoogle ScholarPubMed
Maffe, S., Signorotti, F., Perucca, A., Bielli, M., Hladnik, U., Ragazzoni, E., Maduli, E., Paffoni, P., Dellavesa, P., Paino, A.M., Zenone, F., Parravicini, U., Pardo, N.F., Cucchi, L. & Zanetta, M. (2009). Atypical arrhythmic complications in familial hypokalemic periodic paralysis. Journal of Cardiovascular Medicine 10, 6871.Google Scholar
Masu, M., Iwakabe, H., Tagawa, Y., Miyoshi, T., Yamashita, M., Fukuda, Y., Sasaki, H., Hiroi, K., Nakamura, Y. & Shigemoto, R. (1995). Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene. Cell 80, 757765.Google Scholar
Morgans, C.W., Zhang, J., Jeffrey, B.G., Nelson, S.M., Burke, N.S., Duvoisin, R.M. & Brown, R.L. (2009). TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells. Proceedings of the National Academy of Sciences of the United States of America 106, 1917419178.CrossRefGoogle ScholarPubMed
Mosca, B., Delbono, O., Laura Messi, M., Bergamelli, L., Wang, Z.M., Vukcevic, M., Lopez, R., Treves, S., Nishi, M., Takeshima, H., Paolini, C., Martini, M., Rispoli, G., Protasi, F. & Zorzato, F. (2013). Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice. Nature Communications 4, 1541.Google Scholar
Neuille, M., El Shamieh, S., Orhan, E., Michiels, C., Antonio, A., Lancelot, M.E., Condroyer, C., Bujakowska, K., Poch, O., Sahel, J.A., Audo, I. & Zeitz, C. (2014). Lrit3 deficient mouse (nob6): A novel model of complete congenital stationary night blindness (cCSNB). PLoS One 9, e90342.CrossRefGoogle ScholarPubMed
Peachey, N.S., Ray, T.A., Florijn, R., Rowe, L.B., Sjoerdsma, T., Contreras-Alcantara, S., Baba, K., Tosini, G., Pozdeyev, N., Iuvone, P.M., Bojang, P. Jr., Pearring, J.N., Simonsz, H.J., van Genderen, M., Birch, D.G., Traboulsi, E.I., Dorfman, A., Lopez, I., Ren, H., Goldberg, A.F., Nishina, P.M., Lachapelle, P., McCall, M.A., Koenekoop, R.K., Bergen, A.A., Kamermans, M. & Gregg, R.G. (2012). GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness. American Journal of Human Genetics 90, 331339.Google Scholar
Pearring, J.N., Bojang, P. Jr., Shen, Y., Koike, C., Furukawa, T., Nawy, S. & Gregg, R.G. (2011). A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites. Journal of Neuroscience 31, 1006010066.Google Scholar
Pietri-Rouxel, F., Gentil, C., Vassilopoulos, S., Baas, D., Mouisel, E., Ferry, A., Vignaud, A., Hourde, C., Marty, I., Schaeffer, L., Voit, T. & Garcia, L. (2010). DHPR alpha1S subunit controls skeletal muscle mass and morphogenesis. The EMBO Journal 29, 643654.Google Scholar
Ray, T.A., Heath, K.M., Hasan, N., Noel, J.M., Samuels, I.S., Martemyanov, K.A., Peachey, N.S., McCall, M.A. & Gregg, R.G. (2014). GPR179 is required for high sensitivity of the mGluR6 signaling cascade in depolarizing bipolar cells. Journal of Neuroscience 34, 63346343.Google Scholar
Rios, E. & Pizarro, G. (1991). Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiological Reviews 71, 849908.Google Scholar
Rood, I.M., Deegens, J.K., Merchant, M.L., Tamboer, W.P., Wilkey, D.W., Wetzels, J.F. & Klein, J.B. (2010). Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney International 78, 810816.CrossRefGoogle ScholarPubMed
Sandstrom, R. (2012). UW_RnaSeq_SkMuscle_adult-8wks_C57BL/6. NCBI GEO dataset, NCBI GEO dataset.Google Scholar
Scholl, H.P., Langrova, H., Pusch, C.M., Wissinger, B., Zrenner, E. & Apfelstedt-Sylla, E. (2001). Slow and fast rod ERG pathways in patients with X-linked complete stationary night blindness carrying mutations in the NYX gene. Investigative Ophthalmology & Visual Science 42, 27282736.Google ScholarPubMed
Shen, Y., Heimel, J.A., Kamermans, M., Peachey, N.S., Gregg, R.G. & Nawy, S. (2009). A transient receptor potential-like channel mediates synaptic transmission in rod bipolar cells. Journal of Neuroscience 29, 60886093.CrossRefGoogle ScholarPubMed
Soto, F., Ma, X., Cecil, J.L., Vo, B.Q., Culican, S.M. & Kerschensteiner, D. (2012). Spontaneous activity promotes synapse formation in a cell-type-dependent manner in the developing retina. Journal of Neuroscience 32, 54265439.Google Scholar
Specht, D., Wu, S.B., Turner, P., Dearden, P., Koentgen, F., Wolfrum, U., Maw, M., Brandstatter, J.H. & tom Dieck, S. (2009). Effects of presynaptic mutations on a postsynaptic Cacna1s calcium channel colocalized with mGluR6 at mouse photoreceptor ribbon synapses. Investigative Ophthalmology & Visual Science 50, 505515.Google Scholar
Tummala, S.R., Neinstein, A., Fina, M.E., Dhingra, A. & Vardi, N. (2014). Localization of Cacna1s to ON bipolar dendritic tips requires mGluR6-related cascade elements. Investigative Ophthalmology & Visual Science 55, 14831492.CrossRefGoogle Scholar
van Genderen, M.M., Bijveld, M.M., Claassen, Y.B., Florijn, R.J., Pearring, J.N., Meire, F.M., McCall, M.A., Riemslag, F.C., Gregg, R.G., Bergen, A.A. & Kamermans, M. (2009). Mutations in TRPM1 are a common cause of complete congenital stationary night blindness. American Journal of Human Genetics 85, 730736.Google Scholar
Zeitz, C., Jacobson, S.G., Hamel, C.P., Bujakowska, K., Neuille, M., Orhan, E., Zanlonghi, X., Lancelot, M.E., Michiels, C., Schwartz, S.B., Bocquet, B., Antonio, A., Audier, C., Letexier, M., Saraiva, J.P., Luu, T.D., Sennlaub, F., Nguyen, H., Poch, O., Dollfus, H., Lecompte, O., Kohl, S., Sahel, J.A., Bhattacharya, S.S. & Audo, I. (2013). Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness. American Journal of Human Genetics 92, 6775.CrossRefGoogle ScholarPubMed
Zeitz, C., van Genderen, M., Neidhardt, J., Luhmann, U.F., Hoeben, F., Forster, U., Wycisk, K., Matyas, G., Hoyng, C.B., Riemslag, F., Meire, F., Cremers, F.P. & Berger, W. (2005). Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15 Hz flicker electroretinogram. Investigative Ophthalmology & Visual Science 46, 43284335.Google Scholar