Cilantro leaf harbors a potent potassium channel-activating anticonvulsant

Abstract

Herbs have a long history of use as folk medicine anticonvulsants, yet the underlying mechanisms often remain unknown. Neuronal voltage-gated potassium channel subfamily Q (KCNQ) dysfunction can cause severe epileptic encephalopathies that are resistant to modern anticonvulsants. Here we report that cilantro (Coriandrum sativum), a widely used culinary herb that also exhibits antiepileptic and other therapeutic activities, is a highly potent KCNQ channel activator. Screening of cilantro leaf metabolites revealed that one, the long-chain fatty aldehyde (E)-2-dodecenal, activates multiple KCNQs, including the predominant neuronal isoform, KCNQ2/KCNQ3 [half maximal effective concentration (EC₅₀), 60 ± 20 nM], and the predominant cardiac isoform, KCNQ1 in complexes with the type I transmembrane ancillary subunit (KCNE1) (EC₅₀, 260 ± 100 nM). (E)-2-dodecenal also recapitulated the anticonvulsant action of cilantro, delaying pentylene tetrazole-induced seizures. In silico docking and mutagenesis studies identified the (E)-2-dodecenal binding site, juxtaposed between residues on the KCNQ S5 transmembrane segment and S4-5 linker. The results provide a molecular basis for the therapeutic actions of cilantro and indicate that this ubiquitous culinary herb is surprisingly influential upon clinically important KCNQ channels.-Manville, R. W., Abbott, G. W. Cilantro leaf harbors a potent potassium channel-activating anticonvulsant.

Keywords: KCNQ1; KCNQ2; KCNQ3; epilepsy; herbal medicine.

Figure 1

Cilantro extract differentially activates homomeric KCNQ channels All error bars indicate sem. A) Image of the fresh cilantro (Coriander sativum) used in this study. B) Topological representation of a Kv channel showing 2 of the 4 subunits that comprise a channel. C) Extracellular view of the chimeric KCNQ1/KCNQ3 structural model to highlight the anticonvulsant binding pocket (red, KCNQ3-W265). D) Left, mean TEVC current traces for water-injected Xenopus oocytes in the absence (control) or presence of 1% cilantro extract (n = 5). Dashed line here and throughout indicates the 0 current level. Upper inset: the voltage protocol used here and throughout the study unless otherwise indicated. Center: mean tail current for oocytes on left (n = 5). Right: scatter plot of resting membrane potential (E_M) of water-injected oocytes in the absence (Control) or presence of cilantro extract (n = 5). Statistical analyses by 2-way ANOVA. E) Left: mean TEVC current traces for Xenopus oocytes expressing the KCNQ homomers indicated in the absence (control) or presence of 1% cilantro extract (n = 5–6). Arrow indicates time point at which KCNQ tail currents are measured throughout this study. F) Mean tail current (left) and normalized tail current (G/G_max) (right) vs. prepulse voltage relationships for the traces as in the previous panel (n = 5–6). G) Effects of 1% cilantro extract on E_M of unclamped oocytes expressing the channels as in E (n = 5–6). Statistical analyses by 2-way ANOVA. H) Current-fold increase vs. voltage for the KCNQ isoforms indicated, induced by 1% cilantro extract (n = 5–6). I) Scatter plot showing mean ΔV_{0.5 activation} induced by 1% cilantro extract for the KCNQ isoforms indicated (n = 5–6). Statistical analysis by 2-way ANOVA corrected for multiple comparisons.

Figure 2

Cilantro extract differentially activates heteromeric KCNQ channels All error bars indicate sem. A) Left, mean TEVC current traces for Xenopus oocytes expressing KCNQ2/3 in the absence (control) or presence of 1% cilantro extract (n = 6). B) Mean tail current (left) and normalized tail current (G/G_max) (right) vs. prepulse voltage relationships for the KCNQ2/3 traces as in A (n = 6). C) Effects of 1% cilantro extract on E_M of unclamped oocytes expressing KCNQ2/3 (n = 6). Statistical analysis by 2-way ANOVA. D) Left: mean TEVC current traces for Xenopus oocytes expressing homomeric KCNQ1 or heteromeric KCNQ1-KCNE channels as indicated in the absence (control) or presence of 1% cilantro extract (n = 5–6). E) Mean tail current (left) and normalized tail current (G/G_max) (right) vs. prepulse voltage relationships for the traces as in D (n = 5–6). F) Effects of 1% cilantro extract on E_M of unclamped oocytes expressing the channels indicated in D (n = 5–6). Statistical analysis by 2-way ANOVA. G) Left: mean TEVC current traces for Xenopus oocytes expressing homomeric KCNA1 in the absence (control) or presence of 1% cilantro extract (n = 6). H) Mean tail current (left) and normalized tail current (G/G_max) (right) vs. prepulse voltage relationships for the traces as in G (n = 6). I) Effects of 1% cilantro extract on E_M of unclamped oocytes expressing KCNA1 (n = 6). Statistical analysis by 2-way ANOVA. J) Current-fold increase vs. voltage for the Kv channel isoforms indicated, induced by 1% cilantro extract (n = 5–6). K) Scatter plot showing mean ΔV_{0.5 activation} induced by 1% cilantro extract for the Kv channel isoforms indicated; n = 5–6. Statistical analysis by 2-way ANOVA corrected for multiple comparisons.

Figure 3

(E)-2-dodecenal is the KCNQ2/3-activating metabolite in cilantro All error bars indicate sem. Red box indicates the sole hit, (E)-2-dodecenal. A) Chemical structures (left; red indicates oxygen) and electrostatic surface plots (right; red, negative; blue, positive) of the cilantro compounds screened in this study. B) Mean TEVC current traces showing effects of compounds in A (all 100 µM) on KCNQ2/3 expressed in Xenopus oocytes (n = 5–11). C) Mean tail current (left) and normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in B (n = 5–11). 3,4-DHBA, 3,4-dihydroxybenzoic acid.

Figure 4

(E)-2-dodecenal and cilantro extract exhibit similar KCNQ isoform selectivity and anticonvulsant effects. All error bars indicate sem. A) Mean TEVC current traces showing effects of (E)-2-dodecenal (100 µM) on homomeric KCNQ channels expressed in Xenopus oocytes; n = 5 except for KCNQ2 (n = 3). B) Mean tail current (left) and normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in A; n = 5 except for KCNQ2 (n = 3). C) Mean ΔV_{0.5 activation} induced by (E)-2-dodecenal (100 µM) (scatter plot) vs. mean effects of 1% cilantro extract (single bars indicate means from Figs. 1 and 2) for the homomeric KCNQ isoforms indicated; n = 5 except for KCNQ2 (n = 3). D) (E)-2-dodecenal dose responses for homomeric KCNQ1–5 channels (n = 3–5). E, F) Comparison of effects of 1% cilantro extract (E) vs. (E)-2-dodecenal (100 µM) (F) on KCNQ2/3 activation and deactivation rate vs. voltage; n = 6. G) Mean latency to first PTZ-induced seizure for mice preinjected with PBS (n = 16) vs. (E)-2-dodecenal (2 mg/kg) (n = 20) (left) or PBS (n = 29) vs. tridecanal (20 mg/kg) (n = 15) or (E)-2-dodecenal (20 mg/kg) (n = 15) (right). Statistical analysis was by 2-way ANOVA corrected for multiple comparisons. Gold squares = mean values. H) Mean latency to first PTZ-induced seizure for mice preinjected with PBS (n = 14) or 20 mg/kg (E)-2-dodecenal + 2.5 mg/kg XE991 (n = 13). Statistical analysis was by 2-way ANOVA.

Figure 5

KCNQ2/3 activation by (E)-2-dodecenal requires a conserved S5 tryptophan and S4–5 arginine. All error bars indicate sem. A) (E)-2-dodecenal chemical structure (upper and center) and electrostatic surface potentials (red, electron-dense; blue, electron-poor; green, neutral) (lower and center) calculated and plotted using Jmol. B) Chimeric KCNQ1/KCNQ2 structural model (orange, KCNQ2-R213; red, KCNQ2-W236). C) Topological representation of KCNQ5 showing 2 of the 4 subunits, without domain swapping for clarity. Pentagons, approximate position of KCNQ2-R213 (orange) and KCNQ2-W236 (red); D) View of the (E)-2-dodecenal binding site in KCNQ2 predicted by SwissDock. Green line, predicted H-bond. E) Mean TEVC current traces showing effects of (E)-2-dodecenal (100 µM) on KCNQ2-W236L/KCNQ3-W265L (WL/WL) channels expressed in Xenopus oocytes (n = 5–6). F) Mean tail current (left) and mean normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in E (n = 5–6). G) Mean TEVC current traces showing effects of (E)-2-dodecenal (100 µM) on KCNQ2-R213A/KCNQ3-R242 (RA/RA) channels expressed in Xenopus oocytes (n = 5–6). H) Mean tail current (left) and mean normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in G (n = 5–6). I) Current-fold increase vs. voltage in response to (E)-2-dodecenal (100 µM) of wild-type (Q2/Q3), WL/WL, and RA/RA KCNQ2/3 channels (n = 5–6). J) Scatter plot showing ΔV_{0.5 activation} in response to (E)-2-dodecenal (100 µM) of wild-type (Q2/Q3), WL/WL and RA/RA KCNQ2/3 channels (n = 5–6). K) (E)-2-dodecenal dose response calculated from fold increase in current at −60 mV (left) and ΔV_{0.5 activation} (right) for wild-type (Q2/Q3), WL/WL, and RA/RA KCNQ2/3 channels; n = 5–6.

Figure 6

KCNQ2-R213 is essential for KCNQ2/3 activation by (E)-2-dodecenal. All error bars indicate sem. A) Mean TEVC current traces showing effects of (E)-2-dodecenal (100 µM) on KCNQ2/KCNQ3-W265L (upper) and KCNQ2-W236L/KCNQ3 (lower) channels expressed in Xenopus oocytes (n = 5–6). B) Mean tail currents (left) and mean normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in A (n = 5–6). C) (E)-2-dodecenal dose response calculated from ΔV_{0.5 activation} for wild-type, double-mutant (from Fig. 5), and single-mutant KCNQ2/3 channels as indicated; n = 5–6. D) Mean TEVC current traces showing effects of (E)-2-dodecenal (100 µM) on KCNQ2/KCNQ3-R242A (upper) and KCNQ2-R213A/KCNQ3 (lower) channels expressed in Xenopus oocytes (n = 5). E) Mean tail currents (left) and mean normalized tail currents (G/G_max) (right) vs. prepulse voltage relationships for the traces as in D (n = 5). F) (E)-2-dodecenal dose response calculated from ΔV_{0.5 activation} for wild-type, double-mutant (from Fig. 5), and single-mutant KCNQ2/3 channels as indicated (n = 5).

Figure 7

KCNE1 impinges on the KCNQ1 (E)-2-dodecenal binding site. All error bars indicate sem. For structural models: Pink, KCNE1; pale green, KCNQ1-S4; forest green, KCNQ1-S4/5 linker; orange, KCNQ1-R243; pale blue, (E)-2-dodecenal. A) View of the (E)-2-dodecenal binding site predicted by SwissDock in the KCNQ1 (upper) and KCNQ1/KCNE1 (lower) closed-state models. B) View of the (E)-2-dodecenal binding site predicted by SwissDock in the KCNQ1 (upper) and KCNQ1/KCNE1 (lower) open-state models. C) Close-up view of the (E)-2-dodecenal binding site predicted by SwissDock (from A), highlighting the predicted KCNE1-induced shift in the (E)-2-dodecenal binding pose in the closed-state models. D) Mean TEVC current traces (left) and mean tail current vs. prepulse voltage relationships (right) showing effects of (E)-2-dodecenal (100 µM) on channels indicated (n = 5). E) Mean normalized tail current (G/G_max) vs. prepulse voltage relationships showing effects of (E)-2-dodecenal (100 µM) (dashed lines) on the voltage dependence of activation of wild-type and R243A KCNQ1 (Q1) alone (left) or with KCNE1 (E1) (right); n = 5. Wild-type KCNQ1 data are from Fig 4B. F) (E)-2-dodecenal dose response calculated from ΔV_{0.5 activation} for wild-type and R243A KCNQ1 (Q1) alone (left) or with KCNE1 (E1) (right); n = 5. Wild-type KCNQ1 data are from Fig. 4B.