A novel open channel blocker of GABA-A receptors

Carta, Valentina (2014). A novel open channel blocker of GABA-A receptors. (Dissertation, University of Bern, Graduate School for Cellular and Biomedical Sciences)

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GABA-A receptors are chloride ion channels composed of five subunits,
mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit
isoforms have been identified, with the major receptor type in mammalian adult brain
consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous
sedating and anxiolytic drugs such as benzodiazepines. The currently known
endogenous ligands are GABA, neurosteroids and the endocannabinoid 2-
arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion
channel strictly depend on receptor subunit composition and arrangement. GABA-A
receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and
cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions
with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited
currents elicited by GABA with comparable potency as picrotoxin. This
inhibition showed all characteristics of an open channel block. The GABA-A receptor ion
channel is lined by residues from the M2 membrane-spanning segment. To identify
important residues of the pore involved in the interaction with the blocking molecules
PCCP-, a mutation scan was performed in combination with subsequent analysis of the
expressed mutant proteins using electrophysiological techniques.
In a second project we characterised a light-switchable modulator of GABA-A
receptors based on propofol. It was my responsibility to investigate the switching
kinetics in patch clamp experiments. After its discovery in 1980, propofol has become
the most widely used intravenous general anaesthetic. It is commonly accepted that
the anaesthesia induced by this unusually lipophilic drug mostly results from
potentiation of GABA induced currents. While GABA-A receptors respond to a variety of
ligands, they are normally not sensitive towards light. This light sensitivity could be
indirectly achieved by using modulators that can be optically switched between an
active and an inactive form. We tested an azobenzene derivative of propofol where an
aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed
azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with
electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus
oocytes and HEK-cells.
In the third project we wanted to investigate the functional role of GABA-A
receptors in the liver, and their possible involvement in cell proliferation. GABA-A
receptors are also found in a wide range of peripheral tissues, including parts of the
peripheral nervous system and non-neural tissues such as smooth muscle, the female
reproductive system, liver and several cancer tissues. However their precise function in
non neuronal or cancerous cells is still unknown. For this purpose we investigated
expression, localization and function of the hepatocytes GABA-A receptors in model cell
lines and healthy and cancerous hepatocytes.

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