Calmodulin modulates the Ca 2+ -dependent inactivation and expression level of bovine Ca V 2.2 expressed in HEK293T cells

Figures

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  Fig. 1. Bovine CaV2.2 shows Ca2+-dependent inactivation. HEK293T cells transfected with CaV2.2 were whole-cell patched in voltage-clamp mode and depolarized to various potentials from a holding potential of −70 mV for 250 ms. A. The current traces of a representative cell at different potentials using Ca2+ (middle panel) or Ba2+ (bottom panel) as the charge carrier. The black line indicates the trace obtained at +30 mV. B. The normalized current-voltage relationship. The peak inward current measured using Ca2+ (n = 45) or Ba2+ (n = 9) as the charge carrier at different potentials was normalized to the respective maxima of each single cell and then averaged. C. The current evoked by a step depolarization from −70 to +30 mV for 250 ms was recorded using Ca2+ (red) or Ba2+ (blue) as the charge carrier. The residual current at the end of the depolarization was normalized to the peak current as the R250/Ca2+ or R250/Ba2+. D. The average R250/Ca2+ and R250/Ba2+ of CaV2.2. Data are presented as the mean ± SEM. **, p < 0.01 compared to the Ca2+ group using Student's t-test.

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  Fig. 2. Ca2+-binding-deficient CaM mutants increase the R250/Ca2+. We co-expressed CaV2.2 with CaM and CaM mutants with binding deficiencies at the N-, C-, and both lobes (CaM12, CaM34, and CaM1234, respectively) in HEK293T cells. The currents were recorded at +30 mV using Ca2+ or Ba2+ as the charge carrier, and the R250/Ca2+ and R250/Ba2+ were analyzed. A. Schematic representation of the CaM and CaM mutants. B. Representative normalized current traces (each trace was normalized to the inward peak current) from cells expressing CaM or mutants. C. Average R250/Ca2+ and R250/Ba2+. The numbers on each column refer to the number of cells used in each group. Data are presented as the mean ± SEM and were analyzed by one-way ANOVA with Fisher's post hoc test. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 compared to the same expression group using Ca2+ as the charge carrier. #: p < 0.05 and ##: p < 0.001 compared to the R250/Ba2+ of the CaM group. D. Plot of R250/Ca2+ against I30/Ca2+. The R250/Ca2+ of each cell in Fig. 2C was plotted against the I30/Ca2+. The lines indicated the linear regression of each group.

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  Fig. 3. Mutations in the IF residues of CaV2.2 reduce the current. CaV2.2 with mutations in the IF residues (CaV2.2IF/AF and CaV2.2IF/AA) was expressed in the HEK293T cells. A. Schematic representation of CaV2.2 and the constructs. B. The current-voltage relationship. The cell was whole-cell patched and depolarized for 250 ms from a holding potential of −70 mV to various potentials using Ca2+ as the charge carrier. The peak current recorded at each potential was normalized to the inward maxima of each cell. C. The activation curves. Cells were depolarized for 10 ms from a holding potential of −70 mV to various potentials, and the tail inward current obtained at each potential was normalized to the maxima. D. The I30/Ca2+ of CaV2.2 and the mutants. The current was evoked by a depolarization to +30 mV for 250 ms, and the peak inward current was measured. Data are presented as the mean ± SEM and were analyzed by one-way ANOVA with Fisher's post hoc test. *: p < 0.05 and ***: p < 0.001 compared to the wild-type.

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  Fig. 4. CaM12 increases the R250/Ca2+ of mutated CaV2.2. The conserved IF residues of CaV2.2 were mutated to AF (CaV2.2IF/AF) or AA (CaV2.2IF/AF) and expressed in HEK293T cells. A & B. The representative current traces from cells expressing CaV2.2IF/AF and CaV2.2IF/AA co-expressing CaM and CaM mutants. The patched cell was depolarized to +30 mV from a holding potential of −70 mV for 250 ms using Ca2+ (red traces) or Ba2+ (blue traces) as the charge carrier. C & D. Average R250 of CaV2.2IF/AF and CaV2.2IF/AF, respectively. Data are presented as the mean ± SEM and were analyzed by one-way ANOVA with Fisher's post hoc test. *: p < 0.05 and ***: p < 0.001 compared to the same expression group or as indicated.

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  Fig. 5. CaM interacts with the C-tail fragment of CaV2.2. The lysates from HEK293T cells expressing the intracellular C-tail fragment of the wild-type (CT, 72 kDa) (A–D) or mutant CaV2.2 (CTIF/AA) (E) were used for pulldown assays with GST-tagged CaM and mutants (43 kDa) as the baits. The reaction buffer for the assays had no extra addition (None) or contained 100 μM of Ca2+ (Ca) or 5 μM of EGTA (EGTA). The pulldown fraction was then analyzed by Western blotting using a monoclonal antibody against the C-terminal of CaV2.2 (CT) or CaM.

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  Fig. 6. CaM12 enhances the total expression level of CaV2.2. A. Representative Western blot of CaV2.2 in total lysates. Cell lysates isolated from HEK293T cells with no transfection (None), transfected with T-CaV2.2 only (T-CaV2.2, Mock), or transfected with co-expression of CaM, CaM12, CaM34, and CaM1234 were used for Western blot analysis using antibodies against FLAG and β-actin. B. Normalized total CaV2.2 level. The level of T-CaV2.2 was normalized to the level of β-actin in each sample; the values were then normalized to the value of the CaM group in each independent experiment. C. Representative staining of CaV2.2 after being biotinylated. Membrane proteins were first labeled with biotin and then isolated with avidin. The T-CaV2.2 in the total lysate (Total) and avidin-purified fraction (Biotinylated) was analyzed by an antibody against FLAG. D. The ratio of CaV2.2 in the membrane fraction to the total lysate. Data are presented as the mean ± SEM from at least 3 independent experiments and were analyzed by one-way ANOVA with Fisher's post hoc test. *: p < 0.05 compared to the CaM group.