Fig 1.
Chemical structures of the six major kavalactones found in kava.
Fig 2.
Kavain potentiates GABAARs with no apparent subtype selectivity.
(A) Representative traces demonstrating kavain (10–300 μM) enhancing current elicited by 10 μM GABA (EC3) at α1β2γ2L GABAARs in a concentration-dependent manner. (B) Representative traces of current responses elicited by a maximal concentration of GABA (10 mM), in comparison to 300 μM kavain alone. (C) Top panel, Potentiation of GABA-elicited currents (EC3-7) at α1β2, β2γ2L, αxβ2γ2L (x = 1, 2, 3 and 5) and α1βxγ2L (x = 1, 2 and 3) GABAARs by 300 μM kavain. At α4β2δ GABAARs, the GABA control (1 μM) corresponds to an EC30. Data are presented as mean ± SEM. Numbers in bars indicate the number of experiments. No significant difference was found for kavain potentiation at these receptor subtypes (Tukey’s test; p > 0.05 for all pairwise comparisons). Bottom panel, Superimposed current traces of GABA alone (black) and GABA in combination with 300 μM kavain (red) at the corresponding receptor subtypes. The black bars above the traces indicate duration of drug application.
Table 1.
GABA concentration-response curve parameters at various receptor subtypes derived from curve-fitting procedures.
Fig 3.
Kavain produced greater enhancement of GABA-elicited currents at α4β2δ than at α1β2γ2L GABAARs.
(A) GABA concentration-response curves in the absence (black; n = 10) and presence (red; n = 6) of 300 μM kavain at α1β2γ2L GABAARs. The curve parameters are summarised in Table 1. (B) GABA concentration-response curves in the absence (black; n = 4) and presence (red; n = 4) of 300 μM kavain at α4β2δ GABAARs. The curve parameters are summarised in Table 1. (C) Superimposed current responses of maximal GABA in the absence (black) and presence (red) of 300 μM kavain at α1β2γ2L and α4β2δ GABAARs. (D) The effect of kavain on the maximal GABA current responses at α1β2γ2L (n = 6) and α4β2δ (n = 7) GABAARs was compared using the paired t test, and the significance levels are indicated with n.s. (not significant) and **** (p < 0.0001). Data are presented as mean ± SEM.
Fig 4.
Flumazenil-insensitive kavain potentiation has less-than-additive effect on diazepam action.
Top, Representative traces demonstrating responses to control (10 μM GABA); control and 1 μM diazepam; control, 1 μM diazepam and 10 μM flumazenil; control and 10 μM flumazenil; control and 300 μM kavain; control, 300 μM kavain and 10 μM flumazenil; and control, 300 μM kavain and 1 μM diazepam. Bottom, The modulatory effect of diazepam, flumazenil and kavain at α1β2γ2L GABAARs (n = 5). Kavain potentiation was unchanged in the presence of flumazenil (G + K vs. G + K + F; p > 0.05; paired t test). The combination of kavain and diazepam (G + K + D) resulted in greater potentiation than diazepam (G + D; p < 0.001; paired t test) and kavain (G + K; p < 0.0001; paired t test) alone, but the effect was less than the expected additive modulatory effect (dotted line). Data are normalised to the current responses elicited by 10 μM GABA, and are presented as mean ± SEM.
Fig 5.
Kavain modestly reduced etomidate potentiation, but did not affect the direct activation caused by etomidate at α1β2γ2L GABAARs.
Top, Representative traces of current responses elicited by 10 mM GABA (control); 10 μM GABA; 10 μM GABA and 300 μM kavain; 10 μM GABA and 3 μM etomidate; 10 μM GABA, 300 μM kavain and 3 μM etomidate; 30 μM etomidate; 30 μM etomidate and 300 μM kavain. Bottom, Kavain caused a subtle but significant reduction in etomidate potentiation (G2 + E1 vs. G2 + K + E1; n = 7; p < 0.01; paired t test), but had no effect on etomidate activation (E2 vs. E2 + K; n = 6; p > 0.05; paired t test). Data are normalised to the 10 mM GABA responses, and are presented as mean ± SEM.
Fig 6.
Kavain did not affect propofol potentiation, but modestly reduced propofol activation at α1β2γ2L GABAARs.
Top, Representative traces of current responses to 10 mM GABA (control); 10 μM GABA; 10 μM GABA and 300 μM kavain; 10 μM GABA and 10 μM propofol; 10 μM GABA, 300 μM kavain and 10 μM propofol. Middle, Continuous traces demonstrating two consecutive applications of control (100 μM propofol) followed by the co-application of 300 μM kavain with control; and control. Bottom, Receptor modulation produced by propofol alone (G2 + P) was not significantly different from the combination of kavain and propofol (G2 + K + P; n = 5; p > 0.05; paired t test). The agonist effect of propofol (P2) was significantly reduced in the presence of kavain (P2 + K; n = 5; p < 0.01; paired t test). Data are presented as mean ± SEM.
Fig 7.
The pronounced effect of β3N265M point mutation on etomidate and propofol sensitivity.
(A) Representative traces demonstrating the modulatory effect of 3 μM etomidate and 10 μM propofol on GABA EC3 (left) and the agonist effect of 30 μM etomidate and 100 μM propofol relative to 10 mM GABA (right) at α1β3γ2L and α1β3N265Mγ2L GABAARs. (B) The modulatory and agonist effects of etomidate and propofol were markedly diminished at α1β3N265Mγ2L GABAARs. Data are normalised to current responses elicited by 10 mM GABA, and are presented as mean ± SEM. *** p < 0.001; **** p < 0.0001; unpaired t test (mutant vs. wild-type). Numbers above bars indicate number of experiments. GABA + ETO: GABA EC3 + 3 μM etomidate; ETO: 30 μM etomidate; GABA + PRO: GABA EC3 + 10 μM propofol; PRO: 100 μM propofol.