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Figure 1
Phosphorylation sites in rat and mouse Cav3.1 identified by LC-MS/MS. (a) Immuno-purification of rat Cav3.1. Rat Cav3.1 expressed in HEK-293 cells was immunoprecipitated using anti-Cav3.1 antibody, separated by SDS-PAGE gel, and stained with Coomassie Brilliant Blue. Protein molecular weight standards are shown in the left lane, and Cav3.1 protein band >240 kDa is marked as a box in the right lane. (b) Representative MS/MS spectrum of Cav3.1 phosphopeptide TDSLDVQGLGpSR. The phosphorylation site was assigned to S2026 due to observed neutral loss of phosphoric acid H3PO4 and H2O at m/z 606.81 and mass assignments from beta-eliminated y3, y5, y6, y7, y8, y9, and y10 with neutral loss of phosphoric acid H3PO4. (c) Schematic diagram of phosphosites identified in rat Cav3.1 proteins. The identified phosphosites in rat Cav3.1 in HEK-293 cells are marked as red squares. Additionally, the previously identified phosphosites in mouse brain Cav3.1 are marked as white diamonds. The phosphopeptide sequences including phosphorsites are listed in Table 1. MS, mass spectrometry; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis
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Figure 2
Biophysical properties of rat Cav3.1/WT and phospho-silent mutants. (a) Phosphosites are grouped to 8 regions, which are indicated as boxes in Cav3.1 diagram. The regional phospho-silent mutants were constructed by replacing all phosphosites in each region with Ala residues. (b) Representative Ba2+ current traces of Cav3.1/WT and regional phospho-silent mutants expressed in Xenopus oocytes. Currents were evoked by an I-V protocol consisting of 120 ms step pulses from −70 to +40 mV at 10 mV intervals from a holding potential of −90 mV. Scale bars represent 30 ms and 500 nA. (c) I-V relationship plots of Cav3.1/WT and regional phospho-silent mutants. The peak current amplitude values at various test potentials were averaged and plotted against test potentials (n = 9–19). (d) Maximal peak current amplitudes of Cav3.1/WT and regional phospho-silent mutants. The maximal peak current values at −20 mV test potential are represented as bar graphs (n = 9–19; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.01, **; p < 0.001, ***), and given in Table 2. (e) Voltage-dependent activation and channel availability curves of Cav3.1/WT and regional phospho-silent mutants. Chord conductance values were obtained by dividing current amplitudes by driving forces, followed by normalization being then plotted against test potentials (n = 7–18). The channel availability data of Cav3.1/WT and mutants were obtained from normalized current amplitude values upon −20 mV step pulse following various 10 s prepulses. The availability data were averaged and plotted against prepulse potentials (n = 7–17). The smooth curves are from fitting the average availability data to a Boltzmann equation. The V50 values and slope (k) values of activation and channel availability are given in Table 2 (one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.001, ***). (f, g) Activation and inactivation time constants (τact and τinact) of Cav3.1/WT and NT-Ala mutant. τact and τinact were obtained by fitting current traces with two exponential function using Chebyshev fitting method built in Clampfit program. τact (f) and τinact (g) of Cav3.1/WT and NT-Ala mutant were averaged and plotted against test potentials (n = 9–10; Student's unpaired t-test, p < 0.05, *; p < 0.01, **). Representative Ba2+ current traces of Cav3.1/WT and NT-Ala mutant elicited by a step pulse −20 mV were normalized and then superimposed traces are shown for comparison in the inset of (g). Except for NT-Ala mutant, the τact and τinact of the other regional mutants at most test potentials were similar to those of Cav3.1/WT (Table 2). ANOVA, analysis of variance
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Figure 3
Characterization of the CT phospho-silent mutants uncovers phosphosites crucial for the channel activity of Cav3.1. (a) Representative current traces of Cav3.1/WT and single site phospho-silent mutants at the CT. Currents were evoked with the same I-V protocol. Scale bars represent 30 ms and 500 nA. (b) I-V relationships of Cav3.1/WT and CT Ala mutants. The average peak current amplitudes were plotted against test potentials (n = 8–77). (c) Comparison of maximal peak current amplitudes of Cav3.1/WT and CT phospho-silent mutants. The average maximal current amplitude at test potential of –20 mV are represented as bar graphs (n = 8–77; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05, *; p < 0.01, **; p < 0.001, ***). The maximal peak current values are also given in Table 2. (d) Voltage-dependent activation and channel availability curves of Cav3.1/WT and CT Ala mutants. The smooth curves of voltage-dependent activation (n = 7–66) and channel availability (n = 5–21) were obtained by the same analyses described in Figure 2e. The V50 values and slope (k) values of activation and channel availability are shown in Table 2. CT, C-terminus
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Figure 4
Phosphomimetic mutations of Cav3.1 recover the altered properties of the phospho-silent mutants. (a) Representative current traces of Cav3.1/WT and six phosphomimetic mutants (Cav3.1/S18D, S1924D, S2001D, S2163D, S2166D, and S2189D) expressed in Xenopus oocytes. Robust Ba2+ current traces were evoked by the same I-V protocol. Scale bars represent 30 ms and 500 nA. (b) I-V relationships of Cav3.1/WT and the phosphomimetic mutants. Their average peak current amplitudes were plotted against test potentials (n = 15–21). (c) Maximal peak current amplitudes of Cav3.1/WT and the phosphomimetic mutants. The maximal current amplitude values at –20 mV test potential in Figure 4b are represented as bar graphs (n = 15–21; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05, *; p < 0.01, **; Table 2). (d) Voltage-dependent activation (n = 10–20) and channel availability (n = 7–15) curves of Cav3.1/WT and the phosphomimetic mutants. (e, f) Activation and inactivation time constants (τact and τinact) of Cav3.1/WT and S18D mutant. τact and τinact values were obtained with the same method in Figure 2. Average τact and τinact values are plotted against test potentials (n = 10–14). Superimposed Ba2+ current traces of Cav3.1/WT and S18D mutant at –20 mV test potential are displayed in the inset of (f)
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Figure 5
Protein expression levels of HA-Cav3.1/WT and six phospho-silent mutants. (a) The HA-tagged Cav3.1/WT and six phospho-silent mutants were expressed in Xenopus oocytes, and their expression levels in the membrane were detected by indirect immunofluorescence using anti-HA 1st antibody and FITC conjugated 2nd antibody. Shown are representative fluorescence signals imaged by confocal microscopy. (b) Quantitation of fluorescence signals from HA-Cav3.1/WT and six phospho-silent mutants. Their membrane expression levels are from quantitating fluorescence intensities of confocal images using ImageJ program, being corrected for background fluorescence from uninjected oocytes. The average values (±SEM) of maximal fluorescence intensity were presented as bar graphs (Cav3.1/WT, 40.3 ± 3.3; S18A, 34.7 ± 2.9; S1924A, 29.6 ± 2.4; S2001A, 29.5 ± 1.7; S2163A, 30.4 ± 1.6; S2166A, 30.1 ± 2.6; S2189A, 29.8 ± 1.8; n = 15–23; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05, *; p < 0.01, **). (c) Luminometric quantitation of membrane expression of HA-Cav3.1/WT and 5 phospho-silent mutants. Membrane expression levels of HA-Cav3.1 and HA- phospho-silent mutants were detected by indirect immunoluminescence with anti-HA 1st antibody and HRP conjugated 2nd antibody, followed by incubation in Supersignal-femto-ELISA substrate. Their HRP luminescence signals were measured with an EnSpire luminometer and corrected for the background luminescence from uninjected oocytes. Their normalized luminescence intensity values (RLU, relative luminescence unit) to the luminescence value of Cav3.1/WT are represented as bar graphs (Cav3.1/WT, 1.00 ± 0.12; S18A, 0.91 ± 0.07; S1924A, 0.78 ± 0.18; S2001A, 0.79 ± 0.13; S2163A, 0.84 ± 0.14; S2166A, 0.80 ± 0.13; S2189A, 0.79 ± 0.13; n = 5–6). ANOVA, analysis of variance; HRP, horseradish peroxidase
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Figure 6
Modeling for burst firing in TC neurons regulated by the phosphorylation states of Cav3.1. The two Ser residues at the Cav3.1 NT and CT were found to profoundly affect the activity and/or electrophysiological properties of Cav3.1. The altered properties of Cav3.1 were applied to the reported firing model in TC neurons. (a) Firing simulations in thalamocortical relay neurons with the biophysical properties of Cav3.1/WT, S18A, S18D, S2001A, and S2001D. Current (0.088 nA, 100 ms) was injected to holding potential of –74 mV in a dendrite of the cell model. (b) Rebound burst firing simulation of Cav3.1/WT and mutants. The rebound burst firing was elicited after injection of –0.1 nA current for 150 ms to holding potential of –74 mV in the same model. All the simulation data are presented with the same scales. CT, C-terminus; NT, N-terminus; TC, thalamocortical
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Figure 7
Roles of the Ser/Thr residues at the Cav3.1 I–II loop undetected as phosphosites. (a) The proximal I–II loop sequences of three mammalian isoforms of Cav3.1 and Cav3.2 are aligned, displaying conservation of the three phosphosites detected in Cav3.2. Sources of the sequences are as follows: mouse Cav3.2 (mCav3.2, NM_001163691.1); rat Cav3.2 (rCav3.2, NM_153814.2); human Cav3.2 (hCav3.2, AF051946); mCav3.1 (NM_001.08302); rCav3.1 (NM_001177888); hCav3.1 (AF190860). The phosphosites (S442/S445/T446) found in human Cav3.2 are highlighted with the corresponding residues in the mammalian Cav3.1 and Cav3.2 sequences. (b) Representative Ba2+ current traces of Cav3.1/WT, TM, and QM expressed in Xenopus oocytes elicited by I-V protocol. (c) I-V relationship plots of Cav3.1/WT, TM, and QM. Their average peak current amplitudes were plotted against test potentials. (d) The maximal current amplitude values at –20 mV test potential are represented as bar graphs (n = 10–15; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05; Table 2). (e) Voltage-dependent activation (n = 7–13) and channel availability (n = 6–11) curves. Both activation and steady-state inactivation curves of TM and QM were negatively shifted compared to Cav3.1/WT (one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05, *; p < 0.01, **; p < 0.001, ***; Table 2). Voltage-dependent activation and channel availability curves of NT-Ala mutant in Figure 2e were displayed together for comparison. (f, g) Activation and inactivation time constant (τact and τinact) of Cav3.1/WT, TM, and QM. Activation and inactivation τ values are plotted against test potentials (n = 8–14; one-way ANOVA test combined with Dunnett's multiple comparisons test, p < 0.05, *; p < 0.01, **; p < 0.001, ***). Superimposed current traces at –20 mV test potential are displayed in the inset of (g). ANOVA, analysis of variance
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