A study of subunit selectivity, mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABA(A)  receptors.
OBJECTIVE
Most GABA(A)  receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABA(A)  receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABA(A)  receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABA(A)  receptors.


METHODS
Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABA(A)  receptor isoforms. Effects of DS2 on GABA concentration-response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ(0/0)  mice.


RESULTS
Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABA(A)  receptors > α4β3). For δ-GABA(A)  receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABA(A)  receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ(0/0)  mice.


CONCLUSIONS
DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABA(A)  receptors. The effects of DS2 are mediated by an unknown site leading to GABA(A)  receptor isoform selectivity.
NeuroSearch A/S, Ballerup, Denmark.
M L
ML
Jensen
K A
KA
Wafford
A R
AR
Brown
D
D
Belelli
J J
JJ
Lambert
N R
NR
Mirza
D000494:Allosteric Regulation; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000502:physiology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D021122:Protein Subunits / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000502:physiology*; D011994:Recombinant Proteins; D014982:Xenopus laevis
D021122:Protein Subunits; D011725:Pyridines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins