Molecular moieties masking Ca2+-dependent facilitation of voltage-gated Cav2.2 Ca2+ channels

Figures

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  Figure 1.. CDF modulatory domains in the CTD of Cav2.1 and sequence alignment with analogous regions of Cav2.2. Vertical bars (|), identical residues; colons (:), conservative substitutions; periods (.), nonconservative substitutions. Alignment is with human Cav2.1 and 2.2 sequences (GenBank NM_023035.2, NM_001127222.1, NM_000718.3, and CM000671.2).

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  Figure 2.. The absence of CDF in Cav2.2 is not affected by alternative splicing of exon 37. (A–C) Left, representative ICa and IBa evoked before (P1, gray trace) and after (P2, red trace) a prepulse to 20 mV for Cav2.1 (A) and Cav2.2 variants with exon 37b (B) or exon 37a (C). Current traces were overlaid for comparison. Voltage protocol is shown above. P1 and P2 pulses were 10-ms steps from −80 mV to −5 mV (for ICa) or −10 mV (for IBa) 1 s before and 5 ms after, respectively, a 50-ms prepulse to various voltages. For P2 and P1, tail currents were resolved by repolarization to −60 mV for 2 ms before stepping to −80 mV. Right, the ratio of P2 and P1 tail currents is plotted against prepulse voltages for ICa and IBa. Numbers of cells for ICa and IBa are indicated in Table 1. Data represent mean ± SEM.

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  Figure 3.. Repetitive depolarizations cause CDF for Cav2.1 but not Cav2.2. (A–C) ICa or IBa were evoked by 2-ms steps from −80 mV to 0 mV for ICa or −10 mV for IBa at 100 Hz in cells transfected with Cav2.1 (A) or Cav2.2 containing exon 37b (B) or exon 37a (C). The amplitude of each current was normalized to the first current of the train and plotted against pulse number. For clarity, every fifth point is plotted. Numbers of cells for ICa and IBa are indicated in Table 2. Data represent mean ± SEM.

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  Figure 4.. Deletion of exon 46 does not influence the absence of CDF in Cav2.2. (A–D) As in Fig. 3 except cells transfected with Cav2.2 e37b (A and B) or Cav2.2 e37a (C and D) without exon 46. The intracellular recording solution contained 10 or 0.5 mM EGTA as indicated. Data represent mean ± SEM.

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  Figure 5.. The CTDs of Cav2.1 and Cav2.2 distinguish their abilities to undergo CDF. (A–E) As in Fig. 2 (double-pulse protocol) and Fig. 3 (100-Hz protocol) except cells transfected with Cav2.2 channels with the CTD of Cav2.1 (A–C) or Cav2.1 channels with the CTD of Cav2.2 (D and E). In C, interpulse voltage was −140 mV. Gray line representing strong CDF of Cav2.1 ICa (from Fig. 3 A) is overlaid for comparison. Data represent mean ± SEM.

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  Figure 6.. The proximal CTD containing CDF-regulatory domains in Cav2.1 is not functionally conserved in Cav2.2. (A–D) As in Fig. 5 B except cells transfected with Cav2.2 channels containing proximal (A) or distal (B) CTD or Cav2.1 channels containing proximal (C) or distal (D) CTD of Cav2.2. Data represent mean ± SEM.

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  Figure 7.. EF-hand and pre-IQ-IQ domains are the minimal determinants in the CTD that disable CDF in Cav2.2. (A–F) As in Fig. 5 B except cells transfected with Cav2.2 channels containing EF-hand, pre-IQ-IQ, or CBD of Cav2.1 (A–C) or Cav2.1 channels containing corresponding regions of Cav2.2 (D–F). Data represent mean ± SEM.

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  Figure 8.. Both EF-hand and pre-IQ-IQ domains of Cav2.1 are required to unmask CDF in Cav2.2. (A–D) As in Fig. 5 B except cells transfected with Cav2.2 channels containing pre-IQ-IQ and CBD (A), EF-hand and CBD (B), or EF-hand and pre-IQ-IQ (C) of Cav2.1 or Cav2.2 channels containing EF-hand and pre-IQ-IQ of Cav2.1 (D). Data represent mean ± SEM.

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  Figure 9.. CaM differentially interacts with pre-IQ-IQ and EF-pre-IQ-IQ of Cav2.1 and Cav2.2 in pull-down assays. (A and B) GST or GST-tagged Cav2 proteins were incubated with CaM (2.5 µg), and bound CaM was detected by Western blotting. Ponceau staining indicated the amount of each GST protein in the reactions. In B, the signal intensity corresponding to CaM was normalized to that for the GST-protein. A.U., arbitrary units; n.s., not significant; **, P < 0.001, one-way ANOVA and post hoc Tukey test. Data are representative of four independent experiments. (C–F) As in A and B except that variable amounts of CaM (1–10 µg) were used in the assay. In E and F, data were analyzed by two-way ANOVA. There was a significant difference in results obtained for pre-IQ-IQ (P < 0.01) but not EF-pre-IQ-IQ (P = 0.75) of Cav2.1 and Cav2.2. Data are representative of three independent experiments. Data represent mean ± SEM.