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Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Rougier JS, Albesa M, Syam N, Halet G, Abriel H, Viard P - Pflugers Arch. (2014)

Bottom Line: Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo.Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45.These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Physiology, and Pharmacology, University College London, London, WC1E 6BT, UK.

ABSTRACT
Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

No MeSH data available.


USP2-45 reduces the expression Cav1.2 channels at the plasma membrane. a, b Surface biotinylation assays were performed in control and USP2-45-transfected HEK-293 cells. Steptavidin-covered beads were used to pull down the biotinylated proteins and their binding partners. Western blots (WB) show Cav channels and USP2-45 detected in the whole-cell lysates (a) and recovered by pull down with streptavidin (b) from a same experiment (n = 4). Note that the cytosolic USP2-45 was co-precipitated with the biotinylated fraction in (a), suggesting that this de-ubiquitylase binds to proteins expressed at the plasma membrane. As expected, the cytosolic β2 was co-purified with the biotinylated channels, because of their known interaction with Cav1.2. As a control, another cytosolic protein (actin) was not recovered in the biotinylated fraction, indicating that the membrane integrity of the cells was preserved in our experimental conditions. cBar graphs showing how USP2-45 alters the amount of Cav subunits recovered in pull-down experiments. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. USP2-45 specifically decreased the membrane insertion of Cav1.2, whereas the amount of biotinylated α2δ-1 and the proportion of the cytosolic β2 that co-purified with the biotinylated Cav1.2 channels were unchanged. The number of independent experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control. dBar graphs showing the USP2-45-induced decrease in the amount of total Cav proteins (both biotinylated and non-biotinylated) recovered in the corresponding whole-cell lysates of cells exposed to biotin. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. The decreased amount of Cav proteins detected in whole-cell lysates indicates that USP2-45 downregulated all three Cav1.2, β2 and α2δ-1 subunits. **p < 0.01 and ***p < 0.001 when compared with control
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Fig2: USP2-45 reduces the expression Cav1.2 channels at the plasma membrane. a, b Surface biotinylation assays were performed in control and USP2-45-transfected HEK-293 cells. Steptavidin-covered beads were used to pull down the biotinylated proteins and their binding partners. Western blots (WB) show Cav channels and USP2-45 detected in the whole-cell lysates (a) and recovered by pull down with streptavidin (b) from a same experiment (n = 4). Note that the cytosolic USP2-45 was co-precipitated with the biotinylated fraction in (a), suggesting that this de-ubiquitylase binds to proteins expressed at the plasma membrane. As expected, the cytosolic β2 was co-purified with the biotinylated channels, because of their known interaction with Cav1.2. As a control, another cytosolic protein (actin) was not recovered in the biotinylated fraction, indicating that the membrane integrity of the cells was preserved in our experimental conditions. cBar graphs showing how USP2-45 alters the amount of Cav subunits recovered in pull-down experiments. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. USP2-45 specifically decreased the membrane insertion of Cav1.2, whereas the amount of biotinylated α2δ-1 and the proportion of the cytosolic β2 that co-purified with the biotinylated Cav1.2 channels were unchanged. The number of independent experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control. dBar graphs showing the USP2-45-induced decrease in the amount of total Cav proteins (both biotinylated and non-biotinylated) recovered in the corresponding whole-cell lysates of cells exposed to biotin. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. The decreased amount of Cav proteins detected in whole-cell lysates indicates that USP2-45 downregulated all three Cav1.2, β2 and α2δ-1 subunits. **p < 0.01 and ***p < 0.001 when compared with control

Mentions: A reduction of Cav current density may reflect a decrease in the number of channels trafficked to the plasma membrane. Hence, we examined the effect of USP2-45 on the plasma membrane expression of Cav channels by performing surface biotinylation assays. Figure 2a, c shows that USP2-45 reduced the amount of biotinylated Cav1.2, whereas the surface abundance of α2δ-1 and co-precipitated β2 subunits were not affected. Remarkably, Western blots performed on the corresponding whole-cell lysates showed that USP2-45 decreased the protein amount of all three Cav subunits (Fig. 2b, d). It is noteworthy that the cytosolic β2 is pulled down together with the biotinylated Cav1.2 (Fig. 2a). Interestingly, the amount of coprecipitated β suggested that the proportion of β associated with Cav1.2 may be increased in USP2-45-transfected cells (by 120 ± 32 %, n = 4; p < 0.05 when calculating the ratio of co-precipitated β divided by the amount of biotinylated Cav1.2). The same comparison cannot be made regarding α2δ-1, which is known to traffic to the cell surface independently of Cav1.2 [18, 33]. Overall, these experiments showed that USP2-45 decreases the amount of Cav1.2 channels inserted at the plasma membrane and that this downregulation is correlated with a reduction of all three (pore-forming and auxiliary) Cav proteins.Fig. 2


Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Rougier JS, Albesa M, Syam N, Halet G, Abriel H, Viard P - Pflugers Arch. (2014)

USP2-45 reduces the expression Cav1.2 channels at the plasma membrane. a, b Surface biotinylation assays were performed in control and USP2-45-transfected HEK-293 cells. Steptavidin-covered beads were used to pull down the biotinylated proteins and their binding partners. Western blots (WB) show Cav channels and USP2-45 detected in the whole-cell lysates (a) and recovered by pull down with streptavidin (b) from a same experiment (n = 4). Note that the cytosolic USP2-45 was co-precipitated with the biotinylated fraction in (a), suggesting that this de-ubiquitylase binds to proteins expressed at the plasma membrane. As expected, the cytosolic β2 was co-purified with the biotinylated channels, because of their known interaction with Cav1.2. As a control, another cytosolic protein (actin) was not recovered in the biotinylated fraction, indicating that the membrane integrity of the cells was preserved in our experimental conditions. cBar graphs showing how USP2-45 alters the amount of Cav subunits recovered in pull-down experiments. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. USP2-45 specifically decreased the membrane insertion of Cav1.2, whereas the amount of biotinylated α2δ-1 and the proportion of the cytosolic β2 that co-purified with the biotinylated Cav1.2 channels were unchanged. The number of independent experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control. dBar graphs showing the USP2-45-induced decrease in the amount of total Cav proteins (both biotinylated and non-biotinylated) recovered in the corresponding whole-cell lysates of cells exposed to biotin. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. The decreased amount of Cav proteins detected in whole-cell lysates indicates that USP2-45 downregulated all three Cav1.2, β2 and α2δ-1 subunits. **p < 0.01 and ***p < 0.001 when compared with control
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Related In: Results  -  Collection

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Fig2: USP2-45 reduces the expression Cav1.2 channels at the plasma membrane. a, b Surface biotinylation assays were performed in control and USP2-45-transfected HEK-293 cells. Steptavidin-covered beads were used to pull down the biotinylated proteins and their binding partners. Western blots (WB) show Cav channels and USP2-45 detected in the whole-cell lysates (a) and recovered by pull down with streptavidin (b) from a same experiment (n = 4). Note that the cytosolic USP2-45 was co-precipitated with the biotinylated fraction in (a), suggesting that this de-ubiquitylase binds to proteins expressed at the plasma membrane. As expected, the cytosolic β2 was co-purified with the biotinylated channels, because of their known interaction with Cav1.2. As a control, another cytosolic protein (actin) was not recovered in the biotinylated fraction, indicating that the membrane integrity of the cells was preserved in our experimental conditions. cBar graphs showing how USP2-45 alters the amount of Cav subunits recovered in pull-down experiments. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. USP2-45 specifically decreased the membrane insertion of Cav1.2, whereas the amount of biotinylated α2δ-1 and the proportion of the cytosolic β2 that co-purified with the biotinylated Cav1.2 channels were unchanged. The number of independent experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control. dBar graphs showing the USP2-45-induced decrease in the amount of total Cav proteins (both biotinylated and non-biotinylated) recovered in the corresponding whole-cell lysates of cells exposed to biotin. The effect of USP2-45 was expressed as a percentage of reduction in the intensity of Cav protein bands relative to control. The decreased amount of Cav proteins detected in whole-cell lysates indicates that USP2-45 downregulated all three Cav1.2, β2 and α2δ-1 subunits. **p < 0.01 and ***p < 0.001 when compared with control
Mentions: A reduction of Cav current density may reflect a decrease in the number of channels trafficked to the plasma membrane. Hence, we examined the effect of USP2-45 on the plasma membrane expression of Cav channels by performing surface biotinylation assays. Figure 2a, c shows that USP2-45 reduced the amount of biotinylated Cav1.2, whereas the surface abundance of α2δ-1 and co-precipitated β2 subunits were not affected. Remarkably, Western blots performed on the corresponding whole-cell lysates showed that USP2-45 decreased the protein amount of all three Cav subunits (Fig. 2b, d). It is noteworthy that the cytosolic β2 is pulled down together with the biotinylated Cav1.2 (Fig. 2a). Interestingly, the amount of coprecipitated β suggested that the proportion of β associated with Cav1.2 may be increased in USP2-45-transfected cells (by 120 ± 32 %, n = 4; p < 0.05 when calculating the ratio of co-precipitated β divided by the amount of biotinylated Cav1.2). The same comparison cannot be made regarding α2δ-1, which is known to traffic to the cell surface independently of Cav1.2 [18, 33]. Overall, these experiments showed that USP2-45 decreases the amount of Cav1.2 channels inserted at the plasma membrane and that this downregulation is correlated with a reduction of all three (pore-forming and auxiliary) Cav proteins.Fig. 2

Bottom Line: Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo.Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45.These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Physiology, and Pharmacology, University College London, London, WC1E 6BT, UK.

ABSTRACT
Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

No MeSH data available.