Microtransplantation of membranes from cultured cells to Xenopus oocytes: a method to study neurotransmitter receptors embedded in native lipids

Abstract

The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal alpha 7 nicotinic acetylcholine receptors (alpha 7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal alpha 4 beta 2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells.

Figure 1

Properties of AMPA currents in HEK-GluR1 cells and in oocytes microtransplanted with HEK-GluR1 cell membranes. (A) AMPA-dose/current relation from six HEK-GluR1 cells. Peak currents normalized to I_max of each cell (mean I_max = −5.3 ± 0.8 nA; AMPA, 200 μM). Data represent mean ± SEM. (Inset) Sample current elicited by AMPA (100 μM) and blocked by CNQX (20 μM). In all figures, the solid bars show the timing of drug applications. (B) Current-voltage relation, from potential ramps, −120 mV to +60 mV, applied during the steady-state current induced by AMPA (50 μM). Data normalized to the current at −120 mV (−7.7 ± 1.6 nA; average of four HEK-GluR1 cells). (C) AMPA-dose/current response relation from 10 oocytes injected with HEK-GluR1 cell membranes. Peak currents normalized to I_max of each oocyte (mean I_max = −187 ± 92 nA; AMPA, 200 μM). (Inset) Drug concentrations and horizontal bars are as in A. (D) AMPA-current/voltage relation, from four oocytes injected as in C, fitted to a Boltzmann equation (see Materials and Methods). Data normalized to the currents, −149 ± 50 nA amplitude, elicited by AMPA (50 μM) at −120 mV.

Figure 2

AcCho currents from GH(4)C1-α7 cells compared with those of oocytes injected with GH(4)C1-α7 cell membranes. (A) Dose-current response relation from six GH(4)C1-α7 cells. Peak currents normalized to I_max of each cell (mean I_max = −167 ± 52 pA ;n = 6; AcCho, 200 μM). (Inset Left) Current elicited by 100 μM AcCho; Center, plus _5OHind (1 mM; horizontal empty bar); Right, plus MLA (50 nM). (B) AcCho-current-voltage relation obtained with AcCho (100 μM) at different membrane potentials. Data normalized to the AcCho-current at −90 mV (−45 ± 14 pA), and averaged from three cells. (C) AcCho-dose/current response relation from six oocytes injected with GH(4)C1-α7 cell membranes. Peak currents normalized to normalized to I_max of each oocyte (mean I_max = −30.2 ± 5.3 nA; AcCho, 3 mM). (Inset) As in A. (D) AcCho-current/voltage relation, fitted as in Fig. 1, from four oocytes injected as in C. Data normalized to AcCho currents (−40 ± 7 nA; 1 mM AcCho), at −120 mV. MLA and _5OHind were preapplied for 4 s in A and 20 s in C.

Figure 3

AcCho currents of HEK-α4β2 cells compared with those of oocytes injected with HEK-α4β2 cell membranes. (A) Bimodal AcCho-dose/current relation (n = 6 for each point), best fitted to the sum of two Hill equations (superimposed lines) with the following best fitting parameters: n_H = 0.90, EC₅₀ = 8.3 μM; and n_H = 2.0, EC₅₀ = 221 μM. Data normalized to the I_max of each cell (mean peak current −536 ± 60 pA; n = 18; AcCho, 50 μM). (Inset) Current induced by AcCho (30 μM, horizontal filled bar) and reversibly blocked by DHβE (5 μM). (B) Current-voltage relation. Potential ramp, −120 mV to +60 mV, on the steady-state current induced by AcCho (50 μM). Data normalized to the current at −120 mV (−218 ± 52 pA; four cells). (C) AcCho-dose/current response relation from six oocytes injected with HEK-α4β2 cell membranes. Peak currents, normalized to I_max of each oocyte (mean I_max −405 ± 7 nA; AcCho, 500 μM) and best-fitted with a sum of two Hill equations (superimposed lines), gave: n_H = 1 and EC₅₀ = 21.4 μM; n_H = 2.8 and EC₅₀ = 200 μM, respectively. (Inset) As in A. (D) Current-voltage relation, fitted as in Fig. 2, from four oocytes injected as in C. Data normalized to currents (−331 ± 107 nA) elicited by AcCho (50 μM) at −120 mV.