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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.


Effect of USP de-ubiquitylases on Cav1.2 currents. a Representative whole-cell current traces and b corresponding current-voltage (I-V) relationships in Cav1.2/β2/α2δ-1 channels transfected alone with control (white circle (control)), or with USP2-69 (black square) or USP15 (white square) in tsA-201 cells. The maximal conductance and reversal potential were calculated with the I-V fit described in ‘Methods’. Gmax was specifically decreased in USP2-69 (0.3 ± 0.2 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not in USP15-transfected cells (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-69 (48 ± 3 mV versus 54 ± 3 mV in control; NS) or USP15 (46 ± 3 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-69 or USP15 (*p < 0.05). The number of cells is indicated in parentheses. a Representative whole-cell current traces and b corresponding current-voltage relationships in tsA-201 cells transfected with Cav1.2/β2/α2δ-1 channels alone (white circle (control)), or with USP2-45 (black triangle) or the catalytically inactive mutant USP2-45 C67A (white triangle). Gmax was decreased by USP2-45 (0.4 ± 0.1 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not by USP2-45 C67A (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-45 (44 ± 4 mV versus 54 ± 3 mV in control; NS) or USP2-45 C67A (52 ± 1 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-45 (*p < 0.05) and USP2-45 C67A with USP2-45 (#p < 0.05). In all experiments, 5 mM BaCl2 was used to record Cav currents
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Fig1: Effect of USP de-ubiquitylases on Cav1.2 currents. a Representative whole-cell current traces and b corresponding current-voltage (I-V) relationships in Cav1.2/β2/α2δ-1 channels transfected alone with control (white circle (control)), or with USP2-69 (black square) or USP15 (white square) in tsA-201 cells. The maximal conductance and reversal potential were calculated with the I-V fit described in ‘Methods’. Gmax was specifically decreased in USP2-69 (0.3 ± 0.2 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not in USP15-transfected cells (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-69 (48 ± 3 mV versus 54 ± 3 mV in control; NS) or USP15 (46 ± 3 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-69 or USP15 (*p < 0.05). The number of cells is indicated in parentheses. a Representative whole-cell current traces and b corresponding current-voltage relationships in tsA-201 cells transfected with Cav1.2/β2/α2δ-1 channels alone (white circle (control)), or with USP2-45 (black triangle) or the catalytically inactive mutant USP2-45 C67A (white triangle). Gmax was decreased by USP2-45 (0.4 ± 0.1 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not by USP2-45 C67A (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-45 (44 ± 4 mV versus 54 ± 3 mV in control; NS) or USP2-45 C67A (52 ± 1 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-45 (*p < 0.05) and USP2-45 C67A with USP2-45 (#p < 0.05). In all experiments, 5 mM BaCl2 was used to record Cav currents

Mentions: We expressed Cav1.2 together with the accessory subunits β2 and α2δ-1 in tsA-201 cells and assessed the effect of the two USP2 variants and USP15 on Cav currents. Figure 1 shows that co-expressing either USP2-69 (Fig. 1a, b) or USP2-45 (Fig. 1c, d) reduced whole-cell Cav current densities (respectively by 83 ± 7 %, n = 10; p < 0.05 for USP2-69 at 0 mV; and 74 ± 8 % , n = 19; p < 0.05 for USP2-45 at 0 mV). In contrast, USP15, a related de-ubiquitylase [29], had no effect (Fig. 1a, b), suggesting that Cav channels are selectively regulated by USP2 de-ubiquitylases. The decrease in current amplitude was not caused by a shift in the current-voltage relationship (Fig. 1b, d) or inactivation properties of the channels (not shown). In particular, the midpoint of voltage-dependent activation (V50,act), calculated by fitting the current-voltage relationship as indicated in ‘Methods’, was not significatively altered by USP2-69 (−7 ± 3 mV versus −10 ± 1 mV in control; non-significant; NS) or USP2-45 (−9 ± 4 mV versus −10 ± 1 mV in control; NS), hence the amplitude of the effect of either USP2 splice variant can be directly compared at the same voltage. Interestingly, both USP2-69 and USP2-45 significantly increased the slope factor value (k = −9.1 ± 0.9 in USP2-69-transfected cells versus k = −5.9 ± 0.3 in control; P < 0.01; and k = −9.0 ± 1.1 in USP2-45-transfected cells versus k =−5.9 ± 0.4 in control; P < 0.05) which suggests that de-ubiquitylation of the channels may decrease their voltage-sensitivity. Figure 1c, d shows that the catalytically inactive mutant USP2-45C67A failed to regulate Cav1.2 channels. Current densities recorded in USP2-45C67A-transfected cells were similar to control (control 38 ± 5 pA/pF at 0 mV, n = 16; and USP2C67A 37 ± 12 pA/pF at 0 mV, n = 11; NS), confirming that the effect of USP2-45 is mediated by its de-ubiquitylating activity. Further experiments were conducted with the USP2-45 isoform only.Fig. 1


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)

Effect of USP de-ubiquitylases on Cav1.2 currents. a Representative whole-cell current traces and b corresponding current-voltage (I-V) relationships in Cav1.2/β2/α2δ-1 channels transfected alone with control (white circle (control)), or with USP2-69 (black square) or USP15 (white square) in tsA-201 cells. The maximal conductance and reversal potential were calculated with the I-V fit described in ‘Methods’. Gmax was specifically decreased in USP2-69 (0.3 ± 0.2 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not in USP15-transfected cells (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-69 (48 ± 3 mV versus 54 ± 3 mV in control; NS) or USP15 (46 ± 3 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-69 or USP15 (*p < 0.05). The number of cells is indicated in parentheses. a Representative whole-cell current traces and b corresponding current-voltage relationships in tsA-201 cells transfected with Cav1.2/β2/α2δ-1 channels alone (white circle (control)), or with USP2-45 (black triangle) or the catalytically inactive mutant USP2-45 C67A (white triangle). Gmax was decreased by USP2-45 (0.4 ± 0.1 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not by USP2-45 C67A (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-45 (44 ± 4 mV versus 54 ± 3 mV in control; NS) or USP2-45 C67A (52 ± 1 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-45 (*p < 0.05) and USP2-45 C67A with USP2-45 (#p < 0.05). In all experiments, 5 mM BaCl2 was used to record Cav currents
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Fig1: Effect of USP de-ubiquitylases on Cav1.2 currents. a Representative whole-cell current traces and b corresponding current-voltage (I-V) relationships in Cav1.2/β2/α2δ-1 channels transfected alone with control (white circle (control)), or with USP2-69 (black square) or USP15 (white square) in tsA-201 cells. The maximal conductance and reversal potential were calculated with the I-V fit described in ‘Methods’. Gmax was specifically decreased in USP2-69 (0.3 ± 0.2 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not in USP15-transfected cells (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-69 (48 ± 3 mV versus 54 ± 3 mV in control; NS) or USP15 (46 ± 3 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-69 or USP15 (*p < 0.05). The number of cells is indicated in parentheses. a Representative whole-cell current traces and b corresponding current-voltage relationships in tsA-201 cells transfected with Cav1.2/β2/α2δ-1 channels alone (white circle (control)), or with USP2-45 (black triangle) or the catalytically inactive mutant USP2-45 C67A (white triangle). Gmax was decreased by USP2-45 (0.4 ± 0.1 nS/pF versus 0.8 ± 0.1 nS/pF in control; P < 0.05), but not by USP2-45 C67A (0.8 ± 0.2 nS/pF; NS versus control), whilst Vrev was not modified by either USP2-45 (44 ± 4 mV versus 54 ± 3 mV in control; NS) or USP2-45 C67A (52 ± 1 mV; NS versus control). The values for V50,act and slope factors are indicated in the results section. One-way ANOVA statistical analysis compared control with USP2-45 (*p < 0.05) and USP2-45 C67A with USP2-45 (#p < 0.05). In all experiments, 5 mM BaCl2 was used to record Cav currents
Mentions: We expressed Cav1.2 together with the accessory subunits β2 and α2δ-1 in tsA-201 cells and assessed the effect of the two USP2 variants and USP15 on Cav currents. Figure 1 shows that co-expressing either USP2-69 (Fig. 1a, b) or USP2-45 (Fig. 1c, d) reduced whole-cell Cav current densities (respectively by 83 ± 7 %, n = 10; p < 0.05 for USP2-69 at 0 mV; and 74 ± 8 % , n = 19; p < 0.05 for USP2-45 at 0 mV). In contrast, USP15, a related de-ubiquitylase [29], had no effect (Fig. 1a, b), suggesting that Cav channels are selectively regulated by USP2 de-ubiquitylases. The decrease in current amplitude was not caused by a shift in the current-voltage relationship (Fig. 1b, d) or inactivation properties of the channels (not shown). In particular, the midpoint of voltage-dependent activation (V50,act), calculated by fitting the current-voltage relationship as indicated in ‘Methods’, was not significatively altered by USP2-69 (−7 ± 3 mV versus −10 ± 1 mV in control; non-significant; NS) or USP2-45 (−9 ± 4 mV versus −10 ± 1 mV in control; NS), hence the amplitude of the effect of either USP2 splice variant can be directly compared at the same voltage. Interestingly, both USP2-69 and USP2-45 significantly increased the slope factor value (k = −9.1 ± 0.9 in USP2-69-transfected cells versus k = −5.9 ± 0.3 in control; P < 0.01; and k = −9.0 ± 1.1 in USP2-45-transfected cells versus k =−5.9 ± 0.4 in control; P < 0.05) which suggests that de-ubiquitylation of the channels may decrease their voltage-sensitivity. Figure 1c, d shows that the catalytically inactive mutant USP2-45C67A failed to regulate Cav1.2 channels. Current densities recorded in USP2-45C67A-transfected cells were similar to control (control 38 ± 5 pA/pF at 0 mV, n = 16; and USP2C67A 37 ± 12 pA/pF at 0 mV, n = 11; NS), confirming that the effect of USP2-45 is mediated by its de-ubiquitylating activity. Further experiments were conducted with the USP2-45 isoform only.Fig. 1

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.