Cell membranes isolated from neuronal tissue, carrying neurotransmitter receptors and ion channels, can be easily injected into frog oocytes “microtransplanting” both functional neurotransmitter receptors and ion channels. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have and are still embedded in their natural lipidic environment. A limitation of this approach is mainly due to the fact that cell membranes (crude membranes) are isolated from a nervous tissue containing different type of nerve cells, i.e. neurons and glial cells, expressing receptors that may have different characteristics. To avoid this limitation, we injected oocytes with membrane isolated only from glia (glyosome) and we compared some properties of GABA receptors to the GABA receptors expressed by oocytes injected with crude membrane isolated from the same neuronal tissue (mouse cortex). Briefly, the EC50 and nH obtained from the dose-response curve were similar (EC50=90 mM nH=1.2 n=4, EC50=110 mM nH=1.3 n=9, glyosome and crude membrane respectively) as well as the current-voltage relations (reversal potential close to -20 mV). On the contrary, the desensitization properties showed significant difference. In particular, the GABA-current “run-down” (desensitization) induced by repetitive application of 10s GABA (1 mM, 6 applications every 50s) was slower in glyosome-injected oocytes than in crude membrane-injected oocytes (normalized IGABA fall to 75.4±5% n=53 vs. 44.9±4.4% n=53, at the sixth application). Interestingly, a similar result has been already reported from our group using patch-clamp recording on native slice (Palma et al., PNAS 2004, 101:10183-8).

Desensitization of GABAA receptors expressed in glial cell membranes of mouse neocortex using the Xenopus oocyte microtransplantation expression system is reduced compared to that developing in nerve cells

BERNAREGGI, Annalisa;RUZZIER, FABIO;
2009

Abstract

Cell membranes isolated from neuronal tissue, carrying neurotransmitter receptors and ion channels, can be easily injected into frog oocytes “microtransplanting” both functional neurotransmitter receptors and ion channels. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have and are still embedded in their natural lipidic environment. A limitation of this approach is mainly due to the fact that cell membranes (crude membranes) are isolated from a nervous tissue containing different type of nerve cells, i.e. neurons and glial cells, expressing receptors that may have different characteristics. To avoid this limitation, we injected oocytes with membrane isolated only from glia (glyosome) and we compared some properties of GABA receptors to the GABA receptors expressed by oocytes injected with crude membrane isolated from the same neuronal tissue (mouse cortex). Briefly, the EC50 and nH obtained from the dose-response curve were similar (EC50=90 mM nH=1.2 n=4, EC50=110 mM nH=1.3 n=9, glyosome and crude membrane respectively) as well as the current-voltage relations (reversal potential close to -20 mV). On the contrary, the desensitization properties showed significant difference. In particular, the GABA-current “run-down” (desensitization) induced by repetitive application of 10s GABA (1 mM, 6 applications every 50s) was slower in glyosome-injected oocytes than in crude membrane-injected oocytes (normalized IGABA fall to 75.4±5% n=53 vs. 44.9±4.4% n=53, at the sixth application). Interestingly, a similar result has been already reported from our group using patch-clamp recording on native slice (Palma et al., PNAS 2004, 101:10183-8).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/2708479
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