Article Figures & Data
Figures
- Fig. 1.
CavAb block by diltiazem. (A) Structural formula of diltiazem. (B) Concentration curves for inhibition of CaVAb by diltiazem in the resting state (green) at a holding potential of −120 mV with IC50 = 41 μM, and use-dependent block (black) with IC50 = 10.4 μM. (C) Use-dependent block following a train of depolarizations applied at 1 Hz (20 pulses) with IC50 = 10.4 μM (black). Blue and red curves represent use-dependent block by T206S and T206A mutants with IC50 = 40 and 60 μM, respectively. (D) Current records for control and resting-state block by 50 or 100 μM diltiazem during a single depolarization, and use-dependent block by 10 or 100 μM diltiazem during trains of depolarizations.
- Fig. 2.
Structural basis of CavAb block by diltiazem. (A) Overall structure of CavAb illustrated with each subunit distinctly colored (PD, pore domain; VSD, voltage-sensing domain). (B) A cross-sectional view of CavAb in complex with diltiazem. (C) A close-up view of diltiazem binding at the intracellular side of the selectivity filter. Diltiazem is shown in stick model fitting into an Fo-Fc omit map colored in magenta and contoured at 3σ. Nearby side chains are highlighted and shown as sticks. An arrow is shown to mark the position of the fenestration in CavAb (S6, S6-helix; P, P-helix).
- Fig. 3.
Comparison of CavAb block by diltiazem and verapamil. (A) Side view of CaVAb with diltiazem (sticks in green) bound underneath the selectivity filter. Ca2+ is shown as green spheres. The three calcium-binding sites are indicated by the numbers 1, 2, and 3. Portions of the channel are omitted for clarity. (B) Side view of CaVAb with Br-verapamil (sticks in pink) bound reveals overlap between the binding sites of the PAA drug and diltiazem. (C) Side view of CavAb as in (A and B), with superposition of bound diltiazem (green sticks) and verapamil (pink sticks). (D) Orthogonal view of the central cavity of CavAb, showing the overlapping PAA/BZT-binding sites as if one were standing at the bottom of the central cavity and looking upward at the selectivity filter.
- Fig. 4.
Structural basis for allosteric interactions between diltiazem and dihydropyridines bound to CavAb. (A) Surface representation of CaVAb in complex with diltiazem and amlodipine. (B) Zoom-in view of diltiazem binding at the intracellular side of the selectivity filter. Diltiazem is shown in stick format, along with an Fo-Fc omit map contoured at 3σ. Nearby side chains are highlighted and shown in stick format. (C) Comparison of diltiazem binding to CaVAb alone (green) or in the presence of amlodipine (yellow). Dashed arrows indicate the differences between diltiazem positions. (D) Orthogonal view of (C) with calcium bound to the selectivity filter shown in green spheres.
- Fig. 5.
Structural basis for inhibition of CaVAb by amlodipine and diltiazem in combination. (A) Surface representation of diltiazem and amlodipine bound to CaVAb reveals that these drugs bind to different sides of S6. (B) Overall structure of diltiazem-CavAb-amlodipine shown as ribbons.
- Fig. 6.
Diltiazem binding modifies Ca2+ binding in the selectivity filter of CavAb. (A and B) Comparisons of selectivity filter ions in the presence of diltiazem (left) and diltiazem + amlodipine (right). Selectivity filter residues 175–179 and diltiazem are shown in stick format, with Ca2+ shown as green spheres and hydrogen bonds shown as dashed yellow lines. (C and D) Comparisons of Ca2+ bound in Site 1 between diltiazem (left) and diltiazem + amlodipine (right). Side chains from D178 and N181 are shown in each case, along with Ca2+ (green spheres), hydrogen bonds (dashed yellow lines), and estimated intermolecular distances (dashed gray lines).
Additional Files
Data Supplement
- Supplemental Data -
Supplementary Methods
Supplementary Figure 1 - Electron density to support allosteric conformational changes in drug binding positions and Ca2+ binding sites.
Supplementary Table 1 - Data collection, phasing and refinement statistics
- Supplemental Data -
In this issue
Structure of the Diltiazem Receptor Site on Calcium Channels
