Dibenzoylmethane, a novel β-diketone pore blocker of large-conductance calcium-activated potassium channel

Figures

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  Fig. 1.. Docking setup for dibenzoylmethane (DBM) in the BKCa channel pore. (A) Structures of dibenzoylmethane (DBM) tauto-conformers generated at neutral pH (7.0 ± 2.0) and considered during ligand preparation for molecular docking. The diketo form shown on the left was used for docking and subsequent model preparation. (B) Visualization of the cylindrical region used for analysis of pore hydration and cavity volume during molecular dynamics simulations. Side view (left) and cytoplasmic view (right) are shown. The coordinate axes used to track DBM movement are centered in the middle of the cylinder.

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  Fig. 2.. Dibenzoylmethane (DBM) inhibits BKCa channels in mitochondria and the plasma membrane. (A) Representative single-channel recordings of mitoBKCa channel activity showing concentration-dependent inhibition by DBM at negative and positive membrane potentials. (B) Representative multi-channel recordings of plasma membrane BKCa channel activity showing inhibition by DBM at negative and positive membrane potentials. (C) Concentration–response curves showing the voltage dependence of BKCa channel inhibition by DBM. Data were fitted with the Hill equation (n=2÷6). (D) DBM does not alter the single-channel conductance of BKCa channels (293 pS). Conductance was estimated using only well-resolved single-channel openings recorded in the presence of 80 μM DBM at positive membrane potentials (n=3). Data were fitted with linear regression.

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  Fig. 3.. Dibenzoylmethane (DBM) induces a flickering block of BKCa channels. (A) Representative single-channel recording of BKCa channel activity under control conditions at +60 mV. Closed- and open-time distributions are fitted by single exponentials with time constants of τc = 0.34 ms (closed) and τo = 17.22 ms (open), respectively. (B) Recording in the presence of 100 μM DBM, showing enhanced flickering, with τc = 0.45 ms (closed) and τo = 9.37 ms (open). (C) Recording in the presence of 300 μM DBM, showing further enhancement of the flickering block, with τc = 2.27 ms (closed) and τo = 2.74 ms (open). Dwell time distributions were calculated from 9 sec recordings.

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  Fig. 4.. Dibenzoylmethane (DBM) functionally antagonizes paxilline inhibition of BKCa channels. (A) Representative current traces recorded at ±60 mV under control conditions, after washout, and upon application of 400 nM PAX, 80 μM DBM, and PAX together with increasing concentrations of DBM (40, 80, 200 μM). (B) Normalized open probability (Po) of BKCa channels under the indicated conditions. Excess DBM partially reverses the inhibitory effect of paxilline, consistent with competition for an overlapping or closely related inner-pore binding region. Values are shown as mean ± SD. Welch ANOVA test, p < 0.0032 (n=3).

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  Fig 5.. Modulation of BKCa channel activity by dibenzoylmethane (DBM) and related analogs. (A) Representative single-channel current recordings obtained under control conditions and after application of 100 μM of the indicated compounds: PBD, DBM, T-Ch, or DPE. Traces were recorded at holding potentials of +60 mV and −60 mV. Chemical structures of the tested compounds are shown between the control and drug-treated traces. Dashed lines indicate the closed (c) state. Scale bars: 25 pA, 0.25 s. (B) Concentration–response relationships for inhibition of channel activity at +60 mV (left) and −60 mV (right), expressed as the fraction of activity inhibited relative to control. Individual symbols represent single experiments; lines indicate mean values. PBD is the weakest inhibitor across the tested concentration range. One-way ANOVA test, p < 0.001.

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  Fig. 6.. Dibenzoylmethane (DBM) reduces hydration and alters cavity geometry of the BKCa channel pore. (A) The initial states of the BKCa channel with 1, 2, and 4 dibenzoylmethane (DBM) ligands in the channel pore. View from the cytoplasmic side. K+ ions in the selectivity regions are shown as colored spheres; DBM molecules are shown in ball-and-stick representation. (B) Number of water molecules in the pore during MD simulations of systems containing 0, 1, 2, or 4 DBM molecules, shown as linear plots (left) and violin plots (right). (C) Pore cavity volume during the same simulations, shown as linear plots (left) and violin plots (right). (D) Water density in the pore (number of water molecules divided by pore volume), shown as linear plots (left) and violin plots (right). For panels B–D, data are averaged over three independent simulations per condition. The top row shows simulations without an applied electric field; the bottom row shows simulations with an applied electric field corresponding to a membrane potential of 40 mV. Mean (μ) and standard deviation (σ) values calculated from the final 25% of frames are indicated on the violin plots. Cohen’s d factors are shown between…

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  Fig. 7.. Dynamics and interactions of DBM ligands within the BKCa channel pore. (A) Positions of DBM ligands (calculated by averaged locations of their geometric centers) in relation to the pore center, which is at (0, 0, 0) coordinates. Each case is averaged over 3 MD simulations without an electric field are shown on the left and simulations with an electric field on the right. (B) 2D scatter plot showing distances between geometric centers of phenyl rings: Ph-Ph among dibenzoylmethane (DBM) molecules vs. Ph DBM-Ph315 averaged over 3 simulations conducted for each system (1, 2, and 4 DBM molecules, without and with the electric field). Major maxima of point density are indicated by white triangles. Affiliation of Tyr315 to the particular subunit is indicated by different colors. (C) 2D scatter plot showing distances involving carbonyl oxygen atoms of dibenzoylmethane (DBM): O-O among DBM molecules vs. O-K+ ion averaged over 3 simulations conducted for each system (1, 2, and 4 DBM molecules, without and with the electric field). Major maxima of point density are indicated by white triangles.

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  Fig. 8.. Electric field–driven K+ permeation through the BKCa pore in the presence of dibenzoylmethane (DBM). Time evolution of the axial position (altitude along the membrane normal) of individual K+ ions during MD simulations with an applied electric field. Continuous traces represent ion trajectories within the selectivity filter (above 0 level) and the channel pore (below 0 level within the blue area indicating the pore). The membrane spans −20 Å to +20 Å and is shown in grey. The scattered points indicate ion positions outside the channel. Only trajectories of four K+ ions initially located in the selectivity filter are shown. (A) Representative MD simulation showing trajectories of ions in the BK channel with one DBM molecule present in the pore. Colors indicate different K+ ions. (B) ) Superposition of trajectories from all simulations (three simulations per condition) for systems containing 0, 1, 2, or 4 DBM molecules. Colors indicate the different systems. No K+ escape was observed in simulations containing four DBM molecules.