Article Figures & Data
Figures
- Fig. 1.
Sequence alignment between the KCa3.1, Kv2.1, and KcsA channel pore-forming domains. Transmembrane regions S5 and S6 and the pore P-helix are underlined by black bars and labeled. Positions of residues forming a part of the drug receptor sites discussed in this paper are marked and labeled. Amino acids were colored with the Jalview program (http://www.ebi.ac.uk/∼michele/jalview) (Clamp et al., 2004; Waterhouse et al., 2009) using the Zappo color scheme, where hydrophobic residues (I, L, V, A, and M) are colored pink; aromatic residues (F, W, and Y) are colored orange; positively charged residues (K, R, and H) are colored blue; negatively charged residues (D and E) are colored red; hydrophilic residues (S, T, N, and Q) are colored green; P and G colored magenta; and C is colored yellow.
- Fig. 2.
Kv1.2-based homology model of the KCa3.1 channel pore-forming domain. (A) View of the ribbon representation of the homology model of the KCa3.1 channel pore-forming domain from the extracellular side of the membrane. Each subunit is colored individually. Side chains are shown in stick representation. (B) View of the model shown in (A) from the intracellular side of the membrane. (C) Transmembrane view of the model shown in (A). (D) Surface representation of the transmembrane view of the model shown in (A). The pore has been sliced in the middle to highlight the pore lumen and fenestration regions of the structure.
- Fig. 3.
KcsA-based homology model of the KCa3.1 channel pore-forming domain and comparison of the TRAM-34 position in the KcsA- and the Kv1.2-based models. (A) View of the ribbon representation of the homology model of the KCa3.1 channel pore-forming domain from the extracellular side of the membrane. Each subunit is colored individually. (B) Transmembrane view of the model shown in (A). (C) Surface representations of the transmembrane views of TRAM-34 (black) energy minimized in the KcsA- and Kv1.2-based model. The pore has been sliced in the middle to highlight the pore lumen and fenestration regions of the structure.
- Fig. 4.
TRAM-34 interaction with the KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with TRAM-34. Only three subunits are shown for clarity and each subunit is colored individually. Side chains of T250 and V275 are shown in space-filling representation and labeled. TRAM-34 is shown in ball and stick representation with a transparent molecular surface. Transmembrane segments S5 and S6 are labeled for one of the subunits. (B) Intracellular view of the model shown in (A) with all four subunits shown. (C) Close-up of the transmembrane model shown in (A). (D) Surface representation of the transmembrane view of the model shown in (A). Only three subunits are shown for clarity. T250 and V275 side chain atoms are colored light gray (carbon), white (hydrogen), and red (oxygen). TRAM-34 is colored green.
- Fig. 5.
Interaction of several TRAM-34 derivatives with the KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with senicapoc. Only three subunits are shown for clarity and each subunit is colored individually. T250 and V275 side chain atoms are colored light gray (carbon), white (hydrogen), and red (oxygen). Senicapoc is shown in stick representation with a transparent molecular surface. Hydrogen bonds are shown in pink. (B) Overlay of the lowest-energy TRAM-34 (black) and senicapoc (orange) models. Hydrogen bonds between the NH2 group of senicapoc and two T250 residues are shown in pink. The hydrogen bond between one T250 residue and the pyrazole ring of TRAM-34 is shown in green. (C) Representative binding pose of 1-tritylpyrrolodine (TRAM-7) (shown in stick presentation with a transparent molecular surface). (D) One of two dominant binding poses of TRAM-11. (E) Structures of TRAM-34, senicapoc, TRAM-11, TRAM-7, 2,2,2-tris(4-methoxyphenyl)acetonitrile (TRAM-18), and 1-[tris(4-methoxyphenyl)methyl]-1H-pyrazole (TRAM-19).
- Fig. 6.
NS6180 interaction with the KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with NS6180. Only three subunits are shown for clarity and each subunit is colored individually. Side chains of T250 and V275 are shown in space-filling representation and labeled. NS6180 is shown in stick representation with a transparent molecular surface. Transmembrane segments S5 and S6 are labeled for one of the subunits. (B) Intracellular view of the model shown in (A) with all four subunits shown. (C) Close-up of the transmembrane view of the model shown in (A). (D) Surface representation of the transmembrane view of the model shown in (A). Only three subunits are shown for clarity. T250 and V275 side chain atoms are colored light gray (carbon), white (hydrogen), and red (oxygen). NS6180 is colored green.
- Fig. 7.
Nifedipine interaction with the KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with nifedipine. Only two adjacent subunits are shown for clarity and each subunit is colored individually. Side chains of T212 and V272 are shown in space-filling representation and labeled. Nifedipine is shown in stick representation with a transparent molecular surface. Transmembrane segments S5 and S6 are labeled for one of the subunits. (B) Intracellular view of the model shown in (A) with all four subunits shown. (C) Close-up and 90° rotated transmembrane view of the model shown in (B) with only two subunits shown. Hydrogen bonds are shown in green. (D) Surface representation of the transmembrane view of the model shown in (B). All four subunits are shown. Nifedipine is colored green. (E) Concentration-dependent inhibition of wild-type (WT) and mutant KCa3.1 channels by nifedipine. WT KCa3.1 channels are inhibited by nifedipine with an IC50 concentration of 0.74 ± 0.18 µM (h = 0.82 ± 0.28; n = 5). While the fenestration mutants T212F (IC50 = 2.70 ± 1.98 µM; h = 0.71 ± 0.14; n = 11), V272F (IC50 = 5.86 ± 2.12 µM; h = 0.75 ± 0.06; n = 4), and T212F-V272F (10.28 ± 5.18 µM; h = 1.38 ± 0.58; n = 12) decrease nifedipine block by 4-, 8-, and 14-fold, respectively, the pore double mutant T250S-V275A (IC50 = 0.16 ± 0.15 µM; h = 0.73 ± 0.26; n = 4) does not reduce the nifedipine block. (F–H), Individual current traces showing inhibition of KCa3.1 currents in the presence 1 and 10 µM nifedipine. (I) Concentration-dependent relationship of inhibition for the fenestration mutants by TRAM-34 demonstrating the lack of an effect of the fenestration mutants on the action of this pore-selective blocker: T212F (IC50 = 29.0 ± 14.5 nM; h = 0.76 ± 0.26; n = 5), V272F (IC50 = 23.0 ± 11.5 nM; h = 0.64 ± 0.13; n = 4), and T212F-V272F (IC50 = 28.5 ± 6.3 nM; h = 0.89 ± 0.11; n = 6). Data points are expressed as mean ± S.D.; n = number of independent cells used to construct the concentration-response curves.
- Fig. 8.
(S)-4-Phenyl-pyran interaction with KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with (S)-4-phenyl-pyran (shown in stick representation with a transparent molecular surface). Only three subunits are shown for clarity and each subunit is colored individually. Side chains of T250, V275, and T278 are shown in space-filling representation and labeled. Transmembrane segments S5 and S6 are labeled for one of the subunits. (B) Intracellular view of the model shown in (A) with all four subunits shown. (C) Close-up of the transmembrane view shown in (A). (D) Surface representation of the transmembrane view of the model shown in (A). Only three subunits are shown for clarity. T250, V275, and T278 side chain atoms are colored light gray (carbon), white (hydrogen), and red (oxygen). S-4-phenyl-pyran is colored green. (E) Concentration-dependent inhibition of wild-type (WT) and mutant KCa3.1 channels by 4-phenyl-pyran. WT KCa3.1 is inhibited by 4-phenyl-pyran with an IC50 value of 7.7 ± 4.5 nM (h = 1.20 ± 0.27; n = 5). While the pore mutants T250S (IC50 = 969.9 ± 242.7 nM; h = 0.61 ± 0.16; n = 3), T250S-V275A (IC50 = 4.45 ± 1.63 µM; h = 1.02 ± 0.36; n = 7), and T278F (IC50 = 321.04 ± 130.82 nM; h = 0.97 ± 0.38; n = 11) decrease the 4-phenyl-pyran block by 125-, 575-, and 40-fold, respectively, neither of the fenestration mutants V272F (IC50 = 30.1 ± 2.4 nM; h = 1.65 ± 0.48; n = 3) or T212F-V272F (IC50 = 30.2± 4.5 nM; h = 1.54 ± 0.25; n = 5) significantly reduce the 4-phenyl-pyran block. Data points are expressed as mean ± S.D.; n = number of independent cells used to construct the concentration-response curves. (F–H) Current traces showing examples of inhibition of WT KCa3.1, T212-V272F, and T250S-T275A currents by 4-phenyl-pyran.
- Fig. 9.
(R)-4-Phenyl-pyran interaction with KCa3.1 channel pore. (A) Transmembrane view of the ribbon representation of one of the lowest-energy models of the KCa3.1 channel complex with (R)-4-phenyl-pyran (shown in stick representation). Only three subunits are shown for clarity and each subunit is colored individually. Side chains of T250, V275, and T278 are shown in space-filling representation and labeled. Hydrogen bonds are shown in pink. (B) Close-up view of the model shown in (A).
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Figures -
Supplemental Figure 1 - TRAM-34 docked into the fenestration region of the Kv1.2-based KCa3.1 model
Supplemental Figure 2 - TRAM-34 docked into inner pore of the KcsA-based KCa3.1 model
Supplemental Figure 3 - A, TRAM-11 inhibits WT KCa3.1 and the T278F mutant with equal potency (n = 3)
Synthesis of an exemplary 4-phenyl-pyran (Methyl-5-acetyl-4-(4-chloro-3-(trifluoromethyl)phenyl)-2,6-dimethyl-4H-pyran-3-carboxylate (3)
- PDB file 1
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Nifedipine docked into the fenestration of the Kv1.2-based KCa3.1 pore model
- PDB file 2
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KcsA-based model of KCa3.1 channel pore
- PDB file 3
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TRAM-34 docked into the pore of the Kv1.2-based KCa3.1 pore model
- PDB file 4
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TRAM-34 docked into the pore of the Kv1.2-based KCa3.1 pore model
- Supplemental Figures -
In this issue
Molecular Model of Drug Binding in the KCa3.1 Pore
