Limits...
Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.

Liu X, Kerov V, Haeseleer F, Majumder A, Artemyev N, Baker SA, Lee A - Channels (Austin) (2013)

Bottom Line: Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina.However, after postnatal day 13, many PR ribbons retain the immature morphology.Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics; University of Iowa; Iowa City, IA USA; Departments of Otolaryngology-Head and Neck Surgery, and Neurology; University of Iowa; Iowa City, IA USA.

ABSTRACT
Mutations in the gene encoding Cav 1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav 1.4 channels, there are defects in the development of "ribbon" synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav 1.4 channels. Whether defects in PR synapse development due to altered Cav 1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav 1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav 1.4 activation is either enhanced (Cav 1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav 1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

Show MeSH

Related in: MedlinePlus

Figure 1. Rabbit polyclonal antibodies specifically recognize Cav1.4 in transfected cells and in mouse retina. (A and B) western blot (A) and immunofluorescence (B) of HEK293T cells that were untransfected (U) or transfected with Cav1.2, Cav1.3, or Cav1.4. In (Aand B), antibodies against Cav1.2 (top panels), Cav1.3 (middle panels), or Cav1.4 (bottom panels) were used. (C) Western blot of retinal lysate from WT and Cav1.4 KO mice (50 μg protein/lane) probed with Cav1.4 antibodies. Astericks indicate proteins present in WT but not Cav1.4 KO retina. (D) Immunofluorescent labeling with Cav1.4 and RIBEYE antibodies in retina from WT and Cav1.4 KO mice. Nuclei are stained with Hoechst. Lower panels show high magnification images of double-labeling in the outer plexiform layer (OPL). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 2 μm (B); 10 μm, (D, upper panels); 2 μm (D, lower panels). Results shown are representative of at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4042486&req=5

Figure 1: Figure 1. Rabbit polyclonal antibodies specifically recognize Cav1.4 in transfected cells and in mouse retina. (A and B) western blot (A) and immunofluorescence (B) of HEK293T cells that were untransfected (U) or transfected with Cav1.2, Cav1.3, or Cav1.4. In (Aand B), antibodies against Cav1.2 (top panels), Cav1.3 (middle panels), or Cav1.4 (bottom panels) were used. (C) Western blot of retinal lysate from WT and Cav1.4 KO mice (50 μg protein/lane) probed with Cav1.4 antibodies. Astericks indicate proteins present in WT but not Cav1.4 KO retina. (D) Immunofluorescent labeling with Cav1.4 and RIBEYE antibodies in retina from WT and Cav1.4 KO mice. Nuclei are stained with Hoechst. Lower panels show high magnification images of double-labeling in the outer plexiform layer (OPL). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 2 μm (B); 10 μm, (D, upper panels); 2 μm (D, lower panels). Results shown are representative of at least 3 independent experiments.

Mentions: To characterize the subcellular localization of Cav1.4 during development in the mouse retina, we generated polyclonal antibodies against the cytoplasmic N-terminal domain of the Cav1.4 α1 subunit. Since both Cav1.3 and Cav1.2 are thought to be expressed in cone PRs13 and bipolar neurons,14 respectively, we confirmed that the antibodies detected Cav1.4, but not Cav1.2 or Cav1.3, by western blot and immunofluorescence of transfected HEK293T cells (Fig. 1A and B). To determine the specificity of the antibodies for detecting Cav1.4 in the retina, we performed western blotting of retinal lysates from wild-type (WT) mice and mice lacking expression of functional Cav1.4 channels (Cav1.4 KO) due to excision of exons 14 to 17 of the mouse CACNA1F gene.15 In lysate from WT but not Cav1.4 KO retina (Fig. 1C), the antibodies detected a ~200–250 kDa protein that corresponded to the predicted molecular weight of the Cav1.4 α1 subunit. A protein of higher molecular weight was also detected, which may reflect the reduced electrophoretic mobility of aggregated Cav1.4 protein upon denaturation, as such high-molecular bands were also detected in HEK293T cells transfected with Cav1.4 (Fig. 1A). Additional lower-molecular weight proteins were also detected, but were considered non-specific in that they were seen in both genotypes.


Dysregulation of Ca(v)1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.

Liu X, Kerov V, Haeseleer F, Majumder A, Artemyev N, Baker SA, Lee A - Channels (Austin) (2013)

Figure 1. Rabbit polyclonal antibodies specifically recognize Cav1.4 in transfected cells and in mouse retina. (A and B) western blot (A) and immunofluorescence (B) of HEK293T cells that were untransfected (U) or transfected with Cav1.2, Cav1.3, or Cav1.4. In (Aand B), antibodies against Cav1.2 (top panels), Cav1.3 (middle panels), or Cav1.4 (bottom panels) were used. (C) Western blot of retinal lysate from WT and Cav1.4 KO mice (50 μg protein/lane) probed with Cav1.4 antibodies. Astericks indicate proteins present in WT but not Cav1.4 KO retina. (D) Immunofluorescent labeling with Cav1.4 and RIBEYE antibodies in retina from WT and Cav1.4 KO mice. Nuclei are stained with Hoechst. Lower panels show high magnification images of double-labeling in the outer plexiform layer (OPL). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 2 μm (B); 10 μm, (D, upper panels); 2 μm (D, lower panels). Results shown are representative of at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042486&req=5

Figure 1: Figure 1. Rabbit polyclonal antibodies specifically recognize Cav1.4 in transfected cells and in mouse retina. (A and B) western blot (A) and immunofluorescence (B) of HEK293T cells that were untransfected (U) or transfected with Cav1.2, Cav1.3, or Cav1.4. In (Aand B), antibodies against Cav1.2 (top panels), Cav1.3 (middle panels), or Cav1.4 (bottom panels) were used. (C) Western blot of retinal lysate from WT and Cav1.4 KO mice (50 μg protein/lane) probed with Cav1.4 antibodies. Astericks indicate proteins present in WT but not Cav1.4 KO retina. (D) Immunofluorescent labeling with Cav1.4 and RIBEYE antibodies in retina from WT and Cav1.4 KO mice. Nuclei are stained with Hoechst. Lower panels show high magnification images of double-labeling in the outer plexiform layer (OPL). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 2 μm (B); 10 μm, (D, upper panels); 2 μm (D, lower panels). Results shown are representative of at least 3 independent experiments.
Mentions: To characterize the subcellular localization of Cav1.4 during development in the mouse retina, we generated polyclonal antibodies against the cytoplasmic N-terminal domain of the Cav1.4 α1 subunit. Since both Cav1.3 and Cav1.2 are thought to be expressed in cone PRs13 and bipolar neurons,14 respectively, we confirmed that the antibodies detected Cav1.4, but not Cav1.2 or Cav1.3, by western blot and immunofluorescence of transfected HEK293T cells (Fig. 1A and B). To determine the specificity of the antibodies for detecting Cav1.4 in the retina, we performed western blotting of retinal lysates from wild-type (WT) mice and mice lacking expression of functional Cav1.4 channels (Cav1.4 KO) due to excision of exons 14 to 17 of the mouse CACNA1F gene.15 In lysate from WT but not Cav1.4 KO retina (Fig. 1C), the antibodies detected a ~200–250 kDa protein that corresponded to the predicted molecular weight of the Cav1.4 α1 subunit. A protein of higher molecular weight was also detected, which may reflect the reduced electrophoretic mobility of aggregated Cav1.4 protein upon denaturation, as such high-molecular bands were also detected in HEK293T cells transfected with Cav1.4 (Fig. 1A). Additional lower-molecular weight proteins were also detected, but were considered non-specific in that they were seen in both genotypes.

Bottom Line: Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina.However, after postnatal day 13, many PR ribbons retain the immature morphology.Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics; University of Iowa; Iowa City, IA USA; Departments of Otolaryngology-Head and Neck Surgery, and Neurology; University of Iowa; Iowa City, IA USA.

ABSTRACT
Mutations in the gene encoding Cav 1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav 1.4 channels, there are defects in the development of "ribbon" synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav 1.4 channels. Whether defects in PR synapse development due to altered Cav 1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav 1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav 1.4-selective antibodies, we found that Cav 1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav 1.4 activation is either enhanced (Cav 1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav 1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav 1.4 function for efficient PR synapse maturation, and that dysregulation of Cav 1.4 channels in CSNB2 may have synaptopathic consequences.

Show MeSH
Related in: MedlinePlus