Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity.

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.

Modulation by extracellular pH of GABAA receptors expressed in Xenopus oocytes injected with rat brain mRNA.

Rat brain poly(A)(+) mRNA was injected into Xenopus oocytes. After 72-96 hr, GABA(A) receptors expressed in this heterologous system were studied by perfusion of GABA and recording of GABA evoked chloride current under voltage-clamp conditions. The GABA activated currents were blocked by bicuculline and enhanced by flunitrazepam. Acidic (6.4) extracellular pH (pH(e) ) augmented, whereas basic pH (8.4) decreased the current evoked by 100 microM GABA in the respect of the current evoked at pH 7.4. Concentration-response curves for GABA evoked chloride currents were built at the three pHs. These data showed that acidic pH does not change the EC50 for GABA but it increases significantly I(max) in comparison to pH 7.4. At pH 8.4 there was a significant decrease of EC50 for GABA. However, there was also a very strong decrease of I(max), so that the overall effect at 100 microM GABA was a decrease of GABA activated chloride current in the respect of the one activated at neutral pH. These data may indicate that on average brain GABA(A) receptors are positively modulated by extracellular acidosis. The opposite may occur in extracellular alcalosis.

Immunocytochemical study by two photon fluorescence microscopy of the distribution of GABA(A) receptor subunits in rat cerebellar granule cells in culture.

An immunocytochemical investigation of the expression of alpha(1), alpha(6), beta(2/3), gamma(2) and delta subunits was performed on rat cerebellum granule cells in culture by the two photon microscopy technique. The first four subunits appear to be expressed abundantly in these cells, whereas the delta one seems to be expressed at a lower level. Another major difference in the distribution of these subunits is that whereas alpha(6), beta(2/3) and gamma(2) appear only on plasma membranes alpha(1) and delta are present mainly in the cell bodies cytoplasm. Still another difference was found in that the presence of gamma(2) on neurites is "polarized", preferentially labelling neurites with the appearance of dendrites. The subunits alpha(6) and beta(2/3) appear to label all types of neurites, with beta(2/3) being by far the most heavily expressed subunit type. A final distinct characteristic is that alpha(6) and, even more, gamma(2) appear to accumulate in the cytoplasmic domains immediately below the cone of emergence of neurites. This suggests a conspicuous transport of such subunits from the site of synthesis in the cell body to the site of final expression in the neurites (dendrites and axon terminals).