Saad Hannan

The approach

While α subunits do not express on the cell surface on their, a handful of other subunits including ρ1 subunits (previously termed GABA_C receptors) are able to access the plasma membrane.

By making chimeras of various combinations of α1 (the predominant cortical α subunit) with ρ1 , we indentified two discreet regions on α subunits that retain α subunits in the endoplasmic reticulum.

Unusually, switching the first and third transmembrane domains of α subunits with those ρ1 subunits allowed cell surface expression.

Substituting two single amino acids allows cell surface expression of α subunits

Using site directed mutagenesis, we identified two single amino acids that control plasma membrane expression of α subunits.

Q241 in the first transmembrane domain which forms a part of the neurosteroid binding site on GABA_A receptors when mutated to an tryptophan (Q241W) allows cell surface expression.

A290 in the third transmembrane domain which forms a part of the volatile anaesthetic binding site on GABA_A receptors when mutated to a tryptophan (A290W) also allows cell surface expression.

α subunit homomers form pentameric ion channels when expressed on the cell surface

By introducing a spontaneous gating mutation (L9’S) we found that α homomers can form spontaneously active pentameric ion channels on the plasma membrane.

In addition, by mutating one the interfaces of α subunits to resemble the GABA binding site found on β subunits, we imparted, albeit reduced, GABA sensitivity to α homomers expressed in Xenopus oocytes.