Article date: March 1999
By: Russell A Nicholson, George Lees, Jian Zheng, Bernard Verdon, in Volume 126, Issue 5, pages 1123-1132
12,14‐dichlorodehydroabietic acid (12,14‐Cl2DHA) reduced GABA‐stimulated uptake of 36Cl− into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABAA receptor function.
12,14‐Cl2DHA did not affect the binding of [3H]‐muscimol to brain membranes but displaced specifically bound [3H]‐EBOB. The inhibitory effect on [3H]‐EBOB binding was not reversible. 12,14‐Cl2DHA reduced the availability of [3H]‐EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14‐Cl2DHA did not affect the rate of association of [3H]‐EBOB with its chloride channel receptor, but increased the initial rate of [3H]‐EBOB dissociation.
12,14‐Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14‐Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells.
Ultimately, 12,14‐Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABAA receptor‐chloride channel complex.
The depolarizing block seen with 12,14‐Cl2DHA in amphotericin‐perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials.
Our investigation demonstrates that 12,14‐Cl2DHA blocks GABA‐dependent chloride entry in mammalian brain and operates as a non‐competitive insurmountable GABAA antagonist. The mechanism likely involves either irreversible binding of 12,14‐Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14‐Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel.
12,14‐dichlorodehydroabietic acid (12,14‐Cl2DHA) reduced GABA‐stimulated uptake of 36Cl− into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABAA receptor function.
12,14‐Cl2DHA did not affect the binding of [3H]‐muscimol to brain membranes but displaced specifically bound [3H]‐EBOB. The inhibitory effect on [3H]‐EBOB binding was not reversible. 12,14‐Cl2DHA reduced the availability of [3H]‐EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14‐Cl2DHA did not affect the rate of association of [3H]‐EBOB with its chloride channel receptor, but increased the initial rate of [3H]‐EBOB dissociation.
12,14‐Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14‐Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells.
Ultimately, 12,14‐Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABAA receptor‐chloride channel complex.
The depolarizing block seen with 12,14‐Cl2DHA in amphotericin‐perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials.
Our investigation demonstrates that 12,14‐Cl2DHA blocks GABA‐dependent chloride entry in mammalian brain and operates as a non‐competitive insurmountable GABAA antagonist. The mechanism likely involves either irreversible binding of 12,14‐Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14‐Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel.
British Journal of Pharmacology (1999) 126, 1123–1132; doi:10.1038/sj.bjp.0702419
DOI: 10.1038/sj.bjp.0702419
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