Dataset Information

Characterization of Cav1.4 complexes (?11.4, ?2, and ?2?4) in HEK293T cells and in the retina.

ABSTRACT: In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 ?1 subunit and auxiliary ? and ?2? subunits. Of the four distinct classes of ? and ?2?, ?2 and ?2?4 are thought to co-assemble with Cav1.4 ?1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 ?1, ?2, and ?2?4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of ?2, named here ?2X13, which, along with ?2a, is present in photoreceptor terminals. Cav1.4 ?1, ?2, and ?2?4 were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Cav1.4 ?1 + ?2X13) containing ?2?4 exhibited weaker voltage-dependent activation than those with ?2?1. Moreover, compared with channels (Cav1.4 ?1 + ?2?4) with ?2a, ?2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by ?2a and ?2X13 variants.

SUBMITTER: Lee A

PROVIDER: S-EPMC4340398 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Publications

Characterization of Cav1.4 complexes (α11.4, β2, and α2δ4) in HEK293T cells and in the retina.

Lee Amy A Wang Shiyi S Williams Brittany B Hagen Jussara J Scheetz Todd E TE Haeseleer Françoise F

The Journal of biological chemistry 20141202 3

In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subuni ...[more]

PMID: 25468907

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Project description:The oscillatory expression of Notch signaling in neural progenitors suggests that both repressors and activators of neural fate specification are expressed in the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification, we hypothesized that genes encoding photoreceptor and ganglion cell fate activators would be highly expressed in Notch1 receptor-bearing (Notch1+) progenitors, directing these cells to differentiate into photoreceptors or into ganglion cells when Notch1 activity is diminished. To identify these genes, we used microarray analysis to study expression profiles of whole retinas and isolated from them Notch1+ cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1+ cells, we utilized immunomagnetic cell separation. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of Notch3 signaling in ganglion cell differentiation. Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1+ cells grouped near the same developmental stage retina cluster. At E14, we found higher expression of repressors (Notch1, Hes5) and activators (Dll3, Atoh7, Otx2) of neuronal differentiation in Notch1+ cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1+ cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1+ cells at P0. We also observed that retinas of wild type animals had only 14% (P < 0.05) more ganglion cells compared to Notch3KO mice. Since this number is relatively small and Notch1 has been shown to contribute to ganglion cell fate specification, we suggested that Notch1 signaling may play a more significant role in RGC development than the Notch3 signaling cascade. Finally, our findings suggest that Notch1+ progenitors--since they heavily express both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators--can differentiate into either ganglion cells or photoreceptors.

Dataset Information

Characterization of Cav1.4 complexes (?11.4, ?2, and ?2?4) in HEK293T cells and in the retina.

Publications

Characterization of Cav1.4 complexes (α11.4, β2, and α2δ4) in HEK293T cells and in the retina.

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