Brugia Malayi - Clark Science Center - Smith College

The Effects of Flavonoids on GABA A
Receptor Modulation via
Patch-Clamping Electrophysiology and Tadpole Assays
Salma Bargach
γ-Aminobutyric acid, or GABA, is an amino acid neurotransmitter in the central nervous system. Known as the most prominent
inhibitory neurotransmitter, its effects counteract those of excitatory pathways preventing seizures or death by excitotoxicity.
GABA A
receptors, a type of GABA receptor, are ligand-gated ion channels that consist of five subunits. When GABA binds
to one of the GABA A
receptor subunits, a chloride ion channel opens creating an influx of chloride ions, making the cell more
negative. Each subunit has a unique composition that corresponds to certain pharmacological properties. One of the subunits,
the γ-subunit, has a benzodiazepine-binding site. Benzodiazepines are allosteric modulators of GABA A
receptors, which increase
the frequency at which the chloride channel opens. The effects produced by benzodiazepines are counteracted by flumazenil,
benzodiazepines’ competitive antagonist.
Flavonoids are a polyphenolic compound found in plants, fruits, and vegetables. They are compounds categorized by their
chemical structure, for instance, flavones, flavonoids, flavonols, and others. Some flavonoids can either act as flumazenil sensitive
benzodiazepines or flumazenil insensitive benzodiazepines, resulting in negative or positive modulatoration of GABA receptor
currents. Also their lipophilic characteristic gives them the ability to cross the blood brain barrier and reduce both oxidative stress
and damage from free radicals, an action that has been suggested to reduce and prevent neurodegenerative diseases.
Patch clamp electrophysiology is an electrophysiology technique that allows for the recording from a single ion channel
or whole cell currents. The specific patch clamp technique used was the whole-cell patch clamp. In this technique, a recording
electrode forms a gigaOhm seal on the cell’s membrane. Negative pressure is then used to break through the membrane allowing
for the recording of all the receptors on the cell (hence ‘whole cell’). The cell is held at -50 mV and, when superfused with GABA,
this opens the chloride ion channel and chloride ions flow out of the cell. This net flow of chloride ions is recorded as a current in
picoamps. Different drugs can either positively or negatively modulate this current. Recordings showing a positive modulation are
identified by increased chloride current via GABA A
receptors expressed in HEK (human embryonic kidney) cells.
In the following study, flavinoid-induced modulation of GABA currents was investigated in human embryonic kidney (HEK)
cells that have been stably transfected with α 1
β 3
γ 2
GABA A
receptors. Using patch clamp electrophysiology, this project focused on
flavonoids that have been suggested to be positive modulators of GABA currents. The flavonoids used were baicalien, catechin
hydrate, and naringenin. Each compound was co-applied with a constant GABA concentration, which also acted as a control, to
see if any of the compounds would positively enhance the GABA currents (suggesting a possible anesthetic/sedative effect of the
flavinoid). Alongside the electrophysiology, tadpole sedation/anesthesia assays were run for all three flavonoids and compared to
propofol, a known anesthetic. Tadpoles were used as the animal model because the concentrations used to anesthetise tadpoles has
been shown to be similar to mammalian levels. In the tadpole assay, both baicalien and naringenin acted as an anesthetic at very
high concentrations. (>300µM) Catechin hydrate had no anesthetic effects. The electrophysiology data confirmed that none of the
compounds tested were positive modulators of GABA A
receptors.
Although these three compounds did not enhance the GABA response, natural compounds are still of interest in
neuropharmacology. As a result, in future experiments I hope to pursue natural compounds that have a higher probability of
binding to the benzodiazepine site on the GABA A
receptor in the search for new sedative or anesthetic compounds. (Supported
by the Howard Hughes Medical Institute)
Advisor: Adam Hall
2012
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