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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-induced de-ubiquitylation of Cav1.2 and α2δ-1 subunits. a Western blots showing the reduction of total Cav1.2, β2 and Cavα2δ-1 proteins in HEK-293 whole-cell lysates used for b pull down of ubiquitylated channels using ubiquitin binding GST-S5A (n = 5). Note that USP2-45 appears to reduce further the amount of Cav1.2 and α2δ-1 subunits recovered in the pull-down assay. cBar graph comparing the effect of USP2-45 on the ubiquitylation of the three different Cav subunits. To illustrate that the decrease in ubiquitylation of Cav1.2 and α2δ-1 cannot be solely explained by the concomitant reduction in total Cav proteins, the intensity of each protein bands recovered by pull-down assays (shown in b) was divided by the intensity of the corresponding band in whole-cell lysates (ubiquitylated and non-ubiquitylated proteins shown in a). The data was expressed as a percentage change of this ratio relative to control. USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. The number of experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control
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Fig4: USP2-45-induced de-ubiquitylation of Cav1.2 and α2δ-1 subunits. a Western blots showing the reduction of total Cav1.2, β2 and Cavα2δ-1 proteins in HEK-293 whole-cell lysates used for b pull down of ubiquitylated channels using ubiquitin binding GST-S5A (n = 5). Note that USP2-45 appears to reduce further the amount of Cav1.2 and α2δ-1 subunits recovered in the pull-down assay. cBar graph comparing the effect of USP2-45 on the ubiquitylation of the three different Cav subunits. To illustrate that the decrease in ubiquitylation of Cav1.2 and α2δ-1 cannot be solely explained by the concomitant reduction in total Cav proteins, the intensity of each protein bands recovered by pull-down assays (shown in b) was divided by the intensity of the corresponding band in whole-cell lysates (ubiquitylated and non-ubiquitylated proteins shown in a). The data was expressed as a percentage change of this ratio relative to control. USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. The number of experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control

Mentions: Next, we examined the effect of USP2-45 on Cav ubiquitylation. We immunoprecipitated Cav1.2 and detected its level of ubiquitylation by using an FK2 anti-ubiquitin antibody, which recognizes both mono- and poly-ubiquitinated proteins. We found that USP2-45 reduced the ubiquitylation status of Cav1.2 (Fig. 3a). Figure 3c shows a significant reduction in the ratio of intensity of the signal detected with the FK2 anti-ubiquitin antibody relative to the total amount of immunoprecipitated Cav1.2, suggesting that USP2-45 de-ubiquitylates Cav1.2 subunits. By contrast, USP2-45 did not significantly alter the ubiquitylation status of immunoprecipitated β2 subunits (Fig. 3c). Because of the lack of sensitivity of the α2δ-1 antibody, together with the downregulating effect of USP2-45, the amount of immunoprecipitated subunits was low and detection of ubiquitin on α2δ-1 was too weak to be reliably analysed using the FK2 antibody (not shown). Hence, we used an alternative approach and performed pull-down experiments using GST-S5a fusion proteins, which recognize poly-ubiquitylated proteins [12]. The specificity of the GST-S5a was demonstrated in our previous works [36]. In cells transfected with the channels only, GST-S5A pulled down all three Cav subunits, whereas no signal was recovered in cells transfected with the empty vector pcDNA3.1 only (Fig. 4a). This result is in line with the fact that the three subunits are tonically ubiquitylated in basal conditions [25, 36]. Most importantly, Fig. 4a shows that the amount of Cav1.2 and α2δ-1 subunits recovered with GST-S5a was drastically reduced in cells co-transfected with USP2-45 compared to control cells transfected with the channels only. As expected from the experiments shown in Fig. 2b, d, USP2-45 also decreased the amount of proteins recovered in the corresponding whole-cell lysates (Fig. 4b, d). To correct for the reduction of Cav proteins and determine the relative change in ubiquitylation of each subunit, we calculated the ratio of ubiquitylated versus total (ubiquitylated and non-ubiquitylated) proteins. The effect of USP2-45 was expressed as a percentage of change from the control value (Fig. 4c). USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. Altogether, these results indicate that both Cav1.2 and α2δ-1, but not β2, are regulated by the USP2-45 de-ubiquitylase.Fig. 3


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-induced de-ubiquitylation of Cav1.2 and α2δ-1 subunits. a Western blots showing the reduction of total Cav1.2, β2 and Cavα2δ-1 proteins in HEK-293 whole-cell lysates used for b pull down of ubiquitylated channels using ubiquitin binding GST-S5A (n = 5). Note that USP2-45 appears to reduce further the amount of Cav1.2 and α2δ-1 subunits recovered in the pull-down assay. cBar graph comparing the effect of USP2-45 on the ubiquitylation of the three different Cav subunits. To illustrate that the decrease in ubiquitylation of Cav1.2 and α2δ-1 cannot be solely explained by the concomitant reduction in total Cav proteins, the intensity of each protein bands recovered by pull-down assays (shown in b) was divided by the intensity of the corresponding band in whole-cell lysates (ubiquitylated and non-ubiquitylated proteins shown in a). The data was expressed as a percentage change of this ratio relative to control. USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. The number of experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: USP2-45-induced de-ubiquitylation of Cav1.2 and α2δ-1 subunits. a Western blots showing the reduction of total Cav1.2, β2 and Cavα2δ-1 proteins in HEK-293 whole-cell lysates used for b pull down of ubiquitylated channels using ubiquitin binding GST-S5A (n = 5). Note that USP2-45 appears to reduce further the amount of Cav1.2 and α2δ-1 subunits recovered in the pull-down assay. cBar graph comparing the effect of USP2-45 on the ubiquitylation of the three different Cav subunits. To illustrate that the decrease in ubiquitylation of Cav1.2 and α2δ-1 cannot be solely explained by the concomitant reduction in total Cav proteins, the intensity of each protein bands recovered by pull-down assays (shown in b) was divided by the intensity of the corresponding band in whole-cell lysates (ubiquitylated and non-ubiquitylated proteins shown in a). The data was expressed as a percentage change of this ratio relative to control. USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. The number of experiments is indicated in parentheses. NS non-significant. ***p < 0.001 when compared with control
Mentions: Next, we examined the effect of USP2-45 on Cav ubiquitylation. We immunoprecipitated Cav1.2 and detected its level of ubiquitylation by using an FK2 anti-ubiquitin antibody, which recognizes both mono- and poly-ubiquitinated proteins. We found that USP2-45 reduced the ubiquitylation status of Cav1.2 (Fig. 3a). Figure 3c shows a significant reduction in the ratio of intensity of the signal detected with the FK2 anti-ubiquitin antibody relative to the total amount of immunoprecipitated Cav1.2, suggesting that USP2-45 de-ubiquitylates Cav1.2 subunits. By contrast, USP2-45 did not significantly alter the ubiquitylation status of immunoprecipitated β2 subunits (Fig. 3c). Because of the lack of sensitivity of the α2δ-1 antibody, together with the downregulating effect of USP2-45, the amount of immunoprecipitated subunits was low and detection of ubiquitin on α2δ-1 was too weak to be reliably analysed using the FK2 antibody (not shown). Hence, we used an alternative approach and performed pull-down experiments using GST-S5a fusion proteins, which recognize poly-ubiquitylated proteins [12]. The specificity of the GST-S5a was demonstrated in our previous works [36]. In cells transfected with the channels only, GST-S5A pulled down all three Cav subunits, whereas no signal was recovered in cells transfected with the empty vector pcDNA3.1 only (Fig. 4a). This result is in line with the fact that the three subunits are tonically ubiquitylated in basal conditions [25, 36]. Most importantly, Fig. 4a shows that the amount of Cav1.2 and α2δ-1 subunits recovered with GST-S5a was drastically reduced in cells co-transfected with USP2-45 compared to control cells transfected with the channels only. As expected from the experiments shown in Fig. 2b, d, USP2-45 also decreased the amount of proteins recovered in the corresponding whole-cell lysates (Fig. 4b, d). To correct for the reduction of Cav proteins and determine the relative change in ubiquitylation of each subunit, we calculated the ratio of ubiquitylated versus total (ubiquitylated and non-ubiquitylated) proteins. The effect of USP2-45 was expressed as a percentage of change from the control value (Fig. 4c). USP2-45 significantly decreased the ubiquitylation of Cav1.2 and α2δ-1 but not β2 subunits. Altogether, these results indicate that both Cav1.2 and α2δ-1, but not β2, are regulated by the USP2-45 de-ubiquitylase.Fig. 3

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.