Properties of HEK-expressed a1b Gly receptors microtransplanted to Xenopus oocytes

Mirko Moroni, Paraskevi Krashia, Lucia Sivilotti. UCL, London, United Kingdom.

Glycine receptors are pentameric ligand-gated ion channels that belong to the Cys-Loop superfamily. The most abundant glycine receptor subtype is formed by the α0 and β subunits; it is found at postsynaptic densities in the adult spinal cord, where it mediates fast synaptic inhibition.

A reporter mutation (L9’T) inserted along the permeation pathway has been widely used as a tool to infer the stoichiometry of receptors in the Cys-Loop superfamily. The L9’T mutation produces an increase in agonist potency proportional to the number of mutated subunits present in the receptor. Using this approach we have previously shown that the α0β receptor assembles in a α0000β00 stoichiometry in HEK cells (Burzomato et al.2003)

When the same approach is applied to these receptors expressed in Xenopus oocytes, its results cannot be interpreted in terms of subunit stoichiometry, as glycine sensitivity was similar for receptors containing mutated subunits and for wild-type receptors.

In order to understand the reasons for this discrepancy, we microtransplanted HEK-expressed mutated (L9’T) and wild-type α0β receptors into oocytes (Marsal J et al.1995). Our results show that the shifts, measured as changes in EC50, produced by the reporter mutation in the glycine dose-response curve of microtransplanted receptors are different from the shifts recorded in HEK cells. Furthermore, the partial agonist β-Ala was more efficacious on wild-type α0β receptors recorded in HEK cells (75% of the maximum response to glycine, n = 4) than on the same receptors microtransplanted into oocytes (39%, n = 4). As it is likely that microtransplanted receptors maintain the original subunit composition and (most of) the original post-translational modifications, other explanations have to be considered. The size of the oocyte and the presence of a vitelline membrane mean that there is a thicker unstirred layer for the agonist to diffuse through in order to reach the receptors, making the concentration rise for agonist applications slower in oocytes than in mammalian cells. Furthermore it is possible that the lipid membrane composition differs in the two heterologous systems.

1. Burzomato, V., et al. Receptors Channels, 2003. 9(6): p. 353-61
2. Marsal, J., et al. Proc Natl Acad Sci U S A, 1995. 92(11): p. 5224-8.