Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

Abstract

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl(-) channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to gamma-aminobutyric acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

Figure 1

Diagram of the procedures used to transplant neurotransmitter receptors from the human brain to the oocyte plasma membrane by injecting either brain cell membranes (Upper) or brain mRNA (Lower) into Xenopus oocytes.

Figure 2

Time course of incorporation of GABA receptors after injecting cell membranes from the human temporal neocortex. Each column represents the mean value (± SEM; n = 6; one frog) of the current elicited by GABA (1 mM) at the times indicated. (Inset) Superimposed currents elicited by GABA (1 mM) at the indicated times (h) after injection. In this and subsequent figures the horizontal bars above the records show the timing of drug applications.

Figure 3

Properties of GABA currents in oocytes injected with human brain membranes. (A) GABA dose/current response relation from five oocytes recorded 36–48 h after injection (one donor frog). GABA currents were normalized to their individual maxima: mean = 164 nA; EC₅₀ = 75 ± 3 μM; n_H = 1.5 ± 0.1. (Inset) Sample of current response to GABA (1 mM). (B) GABA current/membrane voltage relation from an oocyte 36–48 h after membrane injection. (Inset) Superimposed GABA currents (200 μM) obtained at the indicated potentials, 24 h after membrane injection. Reversal potential, −22 mV. Note outward rectification.

Figure 4

Antagonism of the GABA current by Bic. Response to GABA (100 μM; Left), fully blocked by Bic (150 μM; Center), and its recovery (Right) 2 min after withdrawal of Bic, in an oocyte injected with human brain membranes. Representative of four experiments.

Figure 5

Potentiation of GABA currents in oocytes 24–48 h after membrane injection. (A) Current response to GABA (100 μM) potentiated by PB (10 μM). (B) Current response to GABA (3 μM), in a different oocyte from the same batch, and greatly potentiated by PB (10 μM) but not by FLU (0.3 μM) applied 30 s before GABA. Traces were recorded at 2-min intervals (from top to bottom). (C) Current response to GABA (100 μM) in another oocyte (same frog), potentiated by FLU (0.3 μM) applied for 30 s before GABA, and even more by PB (10 μM). Records were obtained at 3-min intervals.

Figure 6

Transmitter-activated currents in oocytes injected with membrane vesicles or mRNA derived from the human brain temporal neocortex. (Upper) Currents elicited by indicated concentrations of transmitters, recorded in an oocyte 24 h after membrane injection. (Lower) Transmitter-activated currents elicited in another oocyte from the same frog, 6 days after mRNA injection. All AMPA currents were elicited after a 30-s pretreatment with, and in the presence of, cyclothiazide (20 μM; not shown).