Identification of Novel Targeting Sites of Calcineurin and CaMKII in Human CaV3.2 T-Type Calcium Channel

Figures

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  Figure 1. Amino acid residues on human Cav3.2 dephosphorylated by calcineurin in vitro. (A) MSMS spectrum of a doubly charged ion with m/z 873.41 matched a phosphopeptide belonging to human Cav3.2, spanning residues 2135 to 2149, with phosphorylation on S2137. The fragment ions experiencing a neutral loss are denoted by an asterisk (*). (B) Cav3.2 phosphorylation sites identified in this study. Phosphorylation of residues highlighted in green indicates a decrease, while phosphorylation of residues highlighted in red indicates an increase in response to calcineurin treatment. The phosphorylation sites emphasized in bold italic font were previously unidentified, to our knowledge. The experiment was repeated twice. The numbers of unique ions/matched spectra were 4/8, 1/3, 3/5, 4/8, 4/19, and 2/6 for S2137, S1999, S2222, S2188, S1198, and S1144, respectively. (C) Selective ion chromatograms (XICs) of ions with indicated m/z at specific liquid chromatography (LC) retention times corresponding to the elution times of the identified phosphopeptides in Table 1. XICs of Flag-Cav3.2 treated with or without calcineurin were compared. (D) A decrease in the phosphorylation of a di-phosphorylated…

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  Figure 2. Regulation of Cav3.2 phosphorylation by calcineurin enzyme activity and binding function in HEK293 cells. (A) Effect of calcineurin inhibition on Cav3.2 phosphorylation. HEK293 cells were transfected with Flag-Cav3.2 for 24 h followed by a 24 h inhibition of calcineurin using cyclosporine A (CSA). The experiment was repeated twice. The numbers of unique ions/matched spectra were 3/10 and 2/4 for S2137 and S1198, respectively. (B) Disruption of calcineurin binding function and Cav3.2 phosphorylation. HEK293 cells were transfected with Flag-tagged wildtype Cav3.2 or a calcineurin-binding deficient mutant, 9A-Cav3.2, for 48 h. XICs of peptides with indicated phosphorylation sites were compared. The experiment was repeated twice. The numbers of unique ions/matched spectra were 3/8 and 2/4 for S2137 and S1198, respectively.

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  Figure 3. Identified S2137 as a novel CaMKII phosphorylation site on human Cav3.2. (A) CaMKII triggered a mobility shift in the SDS-PAGE gel for GST-CII, the GST-fusion protein derived from the C-terminus of Cav3.2. Incubation of GST-CII (1 or 2μg) with or without CaMKII was conducted. Molecular weight markers were designated in kD. n = 3 for control and CaMKII. (B) CaMKII increased the ion signal of the phospho-S2137 peptide. Tryptic peptides extracted from GST-CII gel bands were analyzed by LC-MS/MS. An ion with m/z 873.40 was identified as the phospho-S2137 peptide, while another ion with m/z 555.95 corresponded to the unmodified peptide counterpart. The experiment was repeated twice. The numbers of unique ions/matched spectra were 4/12 for S2137. (C) Verification of phospho-S2137 antibody specificity. The specificity of the antibody was confirmed by the signals generated from wild-type or mutant forms of Flag-tagged Cav3.2 expressed in HEK293 cells. n = 3 for each group. (D) Cav3.2 S2137 phosphorylation by CaMKII and dephosphorylation by calcineurin in the full-length Cav3.2 were confirmed using phospho-S2137 antibody. n = 3 for each group.

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  Figure 4. Phosphorylation of S2137 in endogenous Cav3.2 induced by membrane depolarization through KCl stimulation. (A) Alignment of Cav3.2 sequences from human, rat, and mouse around human Cav3.2 S2137. (B) KCl-induced membrane depolarization led to phosphorylation of S2137 in the native Cav3.2 of mouse CAD cells. Cells were stimulated with 50 mM KCl for 5 min. n = 3 for control and KCl.

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  Figure 5. Mutation of S2137 modulated the Cav3.2 calcium channel function. To mimic phosphorylation, S2137 of Cav3.2 was mutated to aspartic acid, denoted as S2137D; and S1198 of Cav3.2 was mutated to glutamic acid, denoted as S1198E. Whole-cell voltage clamp was performed on HEK293 cells expressing either wild-type (WT) or mutant Cav3.2. The calcineurin inhibitor cyclosporine A (CSA, 10 μM) was added to the bath solution. The representative current traces elicited by the above voltage-clamp protocols are shown in (A). The current density–voltage plots are presented in (B). * p < 0.05 in WT vs. S2137D; # p < 0.05 in WT vs. WT + CSA; and & p < 0.05 in WT vs. S1198E + CSA. n = 25, 14, 13, 14, 14, 17 for WT, WT + CSA, S2137D, S2137D + CSA, S1198E, S1198E + CSA. (C) Steady-state inactivation curves and voltage-dependent curves of wild-type and S2137D Cav3.2. (D) Phosphorylation of S1198 and S2137 directly impacted the Cav3.2 calcium current. Whole-cell voltage clamp was performed on HEK293 cells expressing 9A-mutant (n = 14), S1198A in 9A (n = 12), S2137A in 9A (n = 18), or S1198AS2137A in 9A (n = 13). * p < 0.05 compared to Flag-Cav3.2-9A.