Abundant L-type calcium channel Ca(v)1.3 (alpha1D) subunit mRNA is detected in rod photoreceptors of the mouse retina via in situ hybridization.
OBJECTIVE
Mutations in the CACNA1F gene encoding the L-type calcium channel pore-forming Ca(v)1.4 (alpha1F) subunit in humans result in an incomplete form of congenital stationary night blindness (CSNB2) with residual photoreceptor function. It has been postulated that this residual function, at least in part, may be mediated by another L-type calcium channel subunit, Ca(v)1.3 (alpha1D), expressed within cone photoreceptors. However, the expression of the calcium channel Ca(v)1.3 (alpha1D) subunit within photoreceptors remains debatable due to discrepancies among the immunohistochemical studies reported in the literature. In order to get around the innate complications of utilizing unproven antibodies and to shed light on this discussion, we investigated the mRNA expression profile for the Ca(v)1.3 (alpha1D) subunit in the mouse retina.


METHODS
In situ hybridization was performed on wild type mouse retinal sections with two independent sets of digoxigenin-11-UTP-labeled Ca(v)1.3 (alpha1D)-specific sense and antisense cRNA probes. The two probe sets employed correspond to two distinct regions of the Ca(v)1.3 (alpha1D) subunit mRNA, each encoding a different fragment of the Ca(v)1.3 (alpha1D) polypeptide. In situ hybridization of wild type mouse brain sections with these same probes was performed as an additional control for specificity.


RESULTS
Abundant L-type calcium channel Ca(v)1.3 (alpha1D) subunit mRNA expression was confirmed in most cells of the outer nuclear layer using two independent Ca(v)1.3 (alpha1D)-specific antisense cRNA probes, confirming expression in rod photoreceptors. Ca(v)1.3 (alpha1D) mRNA expression was also observed within most cells of the inner nuclear layer and ganglion cell layers using these same antisense cRNA probes. No labeling of tissue was observed using either sense cRNA probe. In situ detection of concentrated Ca(v)1.3 (alpha1D) mRNA expression within the hippocampus and Purkinje and granule cells of the cerebellum of wild type mouse brain with these same probes confirmed specificity of the probes.


CONCLUSIONS
Our finding of expression of the L-type calcium channel Ca(v)1.3 (alpha1D) subunit mRNA in rods substantiates the possibility that this pore-forming subunit may be a competent component of channels mediating the residual photoreceptor responses observed in mutant mice lacking functional Ca(v)1.4 (alpha1F) subunits and in humans with CSNB2. Furthermore, the combined observations of abundant expression of Ca(v)1.3 (alpha1D) mRNA in wild type rods and the large reduction in the transmission of photoreceptor responses in mice lacking Ca(v)1.4 (alpha1F) raises the possibility that Ca(v)1.3 (alpha1D) protein expression levels, localization, or functioning might be concomitantly altered by disruption of the Ca(v)1.4 (alpha1F) subunit in rods. To date, no studies of Ca(v)1.3 (alpha1D) mRNA nor protein expression levels or localization in cacna1f mutant mice or humans with CSNB2 have been published. Our findings warrant such studies to address the abovementioned possibilities. Finally, the observation of Ca(v)1.3 (alpha1D) mRNA expression in multiple retinal cell types suggests the potential for a broader role for this L-type calcium channel subunit in overall functioning of the normal retina than previously appreciated. We therefore suggest that lesions in either the gene encoding the L-type calcium channel Ca(v)1.3 (alpha1D) subunit or other molecules that interact with and regulate it may underlie one or more retinopathies with currently unidentified molecular etiologies.
Xiao
Hailian
H
Neuroscience Institute, Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA 30310, USA.
Chen
Xiaoming
X
Steele
Ernest C
EC
D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D017403:In Situ Hybridization; D051379:Mice; D008810:Mice, Inbred C57BL; D011689:Purkinje Cells / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D014018:Tissue Distribution
C485995:Cacna1d protein, mouse; D020746:Calcium Channels, L-Type; D012333:RNA, Messenger