Anisatin modulation of the gamma-aminobutyric acid receptor-channel in rat dorsal root ganglion neurons

Abstract

1. Anisatin, a toxic, insecticidally active component of Sikimi plant, is known to act on the GABA system. In order to elucidate the mechanism of anisatin interaction with the GABA system, whole-cell and single-channel patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. 2. Repeated co-applications of GABA and anisatin suppressed GABA-induced whole-cell currents with an EC50 of 1.10 microM. No recovery of currents was observed after washout with anisatin-free solution. 3. However, pre-application of anisatin through the bath had no effect on GABA-induced currents. The decay phase of currents was accelerated by anisatin. These results indicate that anisatin suppression of GABA-induced currents requires opening of the channels and is use-dependent. 4. Anisatin suppression of GABA-induced currents was not voltage dependent. 5. Picrotoxinin attenuated anisatin suppression of GABA-induced currents. [3H]-EBOB binding to rat brain membranes was competitively inhibited by anisatin. These data indicated that anisatin bound to the picrotoxinin site. 6. At the single-channel level, anisatin did not alter the open time but prolonged the closed time. The burst duration was reduced and channel openings per burst were decreased indicating that anisatin decreased the probability of openings.

Figure 1

Suppression of GABA-induced currents by co-application of anisatin and GABA, and the structure of anisatin. (a) Current records in response to 5 s application of 30 μM GABA with and without anisatin. GABA alone (solid bar); co-application of GABA and anisatin (broken bar). (b) Time course of changes in peak current amplitude by repeated applications of GABA alone and co-applications of GABA and anisatin (solid line). The peak amplitude of current gradually decreased during repeated co-applications. No recovery was observed after washing with anisatin-free solution.

Figure 2

Concentration-response relationship for the suppression of GABA-induced peak currents by co-application of anisatin. Currents were induced by 5 s applications of 30 μM GABA, and suppressed by anisatin in a concentration-dependent manner with an EC₅₀ of 1.10±1.40 μM. The Hill coefficient was estimated to be 0.75+0.13. Mean±s.d. with the numbers of experiments in parentheses.

Figure 3

Pre-application of anisatin alone does not suppress the 30 μM GABA-induced currents. (a) Current records before and after pre-application of 1 μM anisatin alone for 60 s. (b) Time course of the changes in current amplitude during the experiment shown in (a). The ratios of after (pre-application of anisatin)/before (control) is 104.2±3.2 (n=5).

Figure 4

Prolonged co-application (30 s) of 1 μM anisatin and 30 μM GABA. (a) Currents induced by GABA alone and co-application of GABA and anisatin. Co-application caused accelerated desensitization of current. (b) The time constant of desensitization was shortened by 1 μM anisatin from 24.6±8.6 to 17.9±5.4 s (n=4).

Figure 5

GABA-induced whole-cell current-voltage relationships with 30 μM GABA alone (•) and co-application of 30 μM GABA and 1 μM anisatin (○) at different holding membrane potentials. The reversal potentials in the absence and presence of anisatin were estimated to be −7.5±13.3 and 0.0±12.8 mV, respectively (mean±s.d., n=4). Anisatin suppression of GABA-induced currents is voltage-independent.

Figure 6

The effect of 1 μM anisatin on picrotoxinin suppression of 30 μM GABA-induced currents. Picrotoxinin suppressed GABA-induced currents in a concentration-dependent manner with an EC₅₀ of 0.42±0.04 μM and a Hill coefficient of 0.89±0.05 (n=5). When the concentration of picrotoxinin was low (0.03–0.3 μM), anisatin suppressed the currents to about 50% of the level achieved by picrotoxinin alone. However, at higher concentrations of picrotoxinin (1–10 μM), anisatin did not suppress the current beyond the level achieved by picrotoxinin alone indicating that anisatin and picrotoxinin share a common binding site (n=3–7).

Figure 7

Scatchard plot of [3H]-EBOB binding to rat brain membranes in the absence and presence of 370 nM anisatin.

Figure 8

Single-channel currents induced by application of 10 μM GABA and co-application of 10 μM GABA and 1 μM anisatin to outside-out membrane patches clamped at a membrane potential of −60 mV. Currents were filtered at 1 kHz. (a) Currents induced by 10 μM GABA occurred during brief isolated openings or during longer openings interrupted by short closures or gaps. (b) Currents induced by co-application of GABA and anisatin.

Figure 9

Open time distributions for currents induced by 10 μM GABA and co-application of 10 μM GABA and 1 μM anisatin. The best fit of three exponential functions is shown. Three time constants in GABA and in GABA plus anisatin are given in (a) and (b), respectively. See text for further explanation.

Figure 10

Closed time distributions for currents induced by 10 μM GABA and co-application of 10 μM GABA and 1 μM anisatin. The best fit of four exponential functions is shown. Four time constants in GABA and GABA plus anisatin are give in (a) and (b), respectively. See text for further explanation.

Figure 11

Distributions of burst durations for currents induced by 10 μM GABA and co-application of 10 μM GABA and 1 μM anisatin. The burst was defined as repeated openings separated by a closure no longer than 5 ms. The best fit of three exponential functions is shown. Three time constants in GABA and GABA plus anisatin are given in (a) and (b), respectively. See text for further explanation.