Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

Figures

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  Figure 1.. Cysteine-modifying reagents and substance P inhibit recombinant Cav3.2 currents with a similar mechanism. A, schematic of the Cav3.2 channel; intra- and extracellular cysteines are indicated by spheres. Shown in red are cysteines mutated in this study. An orange square indicates zinc-binding His191. The purple area indicates the high-affinity zinc-binding site. B, example time course of the effects of 2 mm MTSES and 1 mm DTT) applied in the presence of MTSES) on the Ca2+ current recorded from HEK293 cells transiently overexpressing Cav3.2 and NK1 receptors using a perforated patch clamp. The selective T-type channel blocker Z944 (1 μm) was applied at the end of the experiment. Plotted are peak Ca2+ current amplitudes. Periods of drug application are indicated by vertical gray bars. The inset shows example current traces. C, summary of the effects recorded in the experiments exemplified in B. Individual data points are represented by colored circles. Paired data points from the same experiment are depicted in the same color. D, example time course of an experiment similar to that shown in A, but NEM (200 μm) was applied instead of MTSES. E, summary of the effects recorded in the…

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  Figure 2.. MTSES-induced inhibition of Cav3 channels requires an intact high-affinity zinc-binding site. A–C, example time courses showing the effects of MTSES (2 mm), DTT (1 mm, applied in the presence of MTSES), and Z944 (1 μm) on the Ca2+ current recorded from HEK293 cells transiently overexpressing Cav3.2 H191Q (A), WT Cav3.1 (B), or Cav3.1 Q172H (C). Plotted are peak Ca2+ current amplitudes; periods of drug application are indicated by vertical gray bars. The inset shows example current traces. D, summary of the effects recorded in the experiments exemplified in A. Individual data points are represented by colored circles. Paired data points from the same experiment are depicted in the same color. Also shown is the dataset for MTSES-induced inhibition of the WT Cav3.2 (gray bar, circles) taken from Fig. 1C. E, summary of the effects recorded in the experiments exemplified in B and C. In bar charts, asterisks denote a significant difference between the groups indicated by the line connectors. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (paired or unpaired t test or one-way ANOVA, as appropriate). Error bars represent mean ± S.E. The number of individual recordings is shown within the bars.

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  Figure 3.. MTSES-induced inhibition of Cav3 channels is reversed by zinc chelation. A and B, example time courses showing the effects of MTSES (2 mm), TPEN (10 μm, applied in the presence of MTSES), and Z944 (1 μm) on the Ca2+ currents recorded from HEK293 cells transiently overexpressing WT Cav3.2 (A) or Cav3.2 H191Q (B). C, summary of the effects recorded in experiments exemplified in A and B. Individual data points are represented by colored circles. Paired data points from the same experiment are depicted in the same color. D and E, experiments similar to those shown in A and B, but MTSES was applied after (and in the presence of) TPEN. F, summary of the effects recorded in the experiments exemplified in D and E. G and H, experiments similar to those shown in D and E, but WT Cav3.1 (G) and Cav3.1 Q172H (H) were investigated. I, summary of the effects recorded in the experiments exemplified in G and H. In bar/scatter charts, asterisks denote a significant difference between the groups indicated by the line connectors. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (paired or unpaired t test or one-way ANOVA, as appropriate). Error bars represent mean ± S.E. The number of…

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  Figure 4.. Homology modeling of the redox/metal-regulatory module in Cav3.2. The initial structure of the human Cav3.2 channel was obtained by homology modeling (see Fig. S1 for further details) based on the structure of human Cav3.1 (35). A, local quality estimate of the model obtained with QMEANBrane. B–D, the redox/metal-regulatory module shown at various rotation points (the protein is shown as ribbons, and key amino acids are indicated). E, electrostatic surface potential of the redox/metal-regulatory module (−50 kT/e to 50 kT/e, in a vacuum).

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  Figure 5.. Extracellular cysteines in the IS1–IS2 loop are necessary for MTSES-mediated inhibition of Cav3.2. A–E, example time courses showing the effects of MTSES (2 mm) and Z944 (1 μm) on the Ca2+ current recorded from HEK293 cells transiently overexpressing Cav3.2 C114A (A), Cav3.2 C123A (B), Cav3.2 C128A (C), and Cav3.2 C133A (D) or a quadruple mutant with Cys-to-Ala mutations at positions 114, 123, 128, and 133 (4xC-A, E). F, summary of the effects recorded in the experiments exemplified in A–E. Individual data points are represented by circles. Asterisks denote a significant difference between the groups indicated by the line connectors. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way ANOVA). Error bars represent mean ± S.E. The number of individual recordings is given below each bar.

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  Figure 6.. Extracellular cysteines in the IS1–IS2 loop are necessary for SP-mediated inhibition of Cav3.2. A, example time courses showing the effects of S9SP (1 μm), DTT (1 mm, applied in the presence of S9SP), and Z944 (1 μm) on the Ca2+ current recorded from HEK293 cells transiently overexpressing Cav3.2 C165A. B, summary of the effects recorded in experiments exemplified in A for HEK293 cells overexpressing Cav3.2 C114A, Cav3.2 C123A, Cav3.2 C128A, Cav3.2 C133A, or Cav3.2 C165A. Individual data points are represented by circles. Asterisks denote significant difference between the groups indicated by the line connectors. *, p < 0.05; ***, p < 0.001 (one-way ANOVA). Error bars represent mean ± S.E. The number of individual recordings is given below each bar.