Recombinant GABA(C) receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human rho1 subunit

Abstract

1. A recombinant adenovirus was generated with the human rho1 GABA(C) receptor subunit (adeno-rho). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant rho1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2. Standard whole-cell recording and Western blot analysis using rho1 GABA(C) receptor antibodies revealed that recombinant rho1 receptors were expressed in HEK293 and 911 cells after adeno-rho infection and exhibited properties similar to those of rho1 receptors after standard transfection. 3. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABA(C)-like current. After adeno-rho infection, however, a GABA(C)-like current appeared in 70-90 % of the neurons. 4. Five days after infection, expression of GABA(C) receptors in hippocampal neurons significantly decreased native GABA(A) receptor currents from 1200 +/- 300 to 150 +/- 70 pA (n = 10). The native glutamate-activated current was unchanged. 5. Hippocampal slices (P8) did not show a native GABA(C)-like current, although recombinant rho1 receptors could be expressed in cultured hippocampal slices after adeno-rho infection. 6. These data indicate that an adenovirus can be used to express recombinant GABA(C) receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases.

Figure 1. Properties of recombinant ρ1 receptors expressed in HEK293 cells after adeno-ρ infection

A, Western blot with N-terminal ρ1 GABA_C receptor antibodies, which recognized a bacterially synthesized N-terminal fusion protein. The band indicated by the asterisk probably represents non-specific staining of an unidentified protein. B, Western blot from uninfected HEK293 cells (lane 1) and HEK293 cells infected with adeno-ρ (2 and 10 PFU cell⁻¹, lanes 2 and 3, respectively). Note the specific band of the ρ1 subunit of the GABA_C receptor (50 kDa) in lanes 2 and 3. C, whole-cell current evoked at a holding potential of −50 mV with GABA (10 μm) in the presence of bicuculline (30 μm) from HEK293 cells after adeno-ρ infection. Decay of the current upon GABA removal was well described by a single exponential component with a time constant (τ) of 7 s. D, mean dose-response relationship for GABA-activated current fitted with a Hill equation (n = 4).

Figure 2. Characteristics of ligand-activated current from hippocampal neurons in culture before and after adeno-ρ infection

Ai-iii, examples of ligand-activated currents from uninfected neurons at a holding potential of −50 mV. Note that GABA (10 μm) in the presence of bicuculline (Bicuc; 30 μm) did not activate current from group i and ii neurons, but evoked a small current from group iii neurons. B, 3-APA (300 μm) did not block the bicuculline-insensitive current from neurons in group iii. SCH 50911 (SCH; 50 μm) was present to block GABA_B receptors. C, the bicuculline-insensitive current from group iii neurons had a linear current-voltage relationship. D, examples of ligand-activated currents from neurons after adeno-ρ infection. Note that GABA (10 μm) in the presence of bicuculline (30 μm) evoked a GABA_C-like current. E, 3-APA (300 μm) completely and reversibly blocked the bicuculline-insensitive current that appeared after adeno-ρ infection. F, dose-response relationship of bicuculline-insensitive current after adeno-ρ infection. Glyc, glycine; Glut, glutamate.

Figure 3. Level and time course of expression of ρ1 GABA_C receptors in neurons and in HEK293 and 911 cell lines after adeno-ρ infection

A, cultured neurons were infected with adeno-GFP and visualized with the red fluorescent dye Ro31-8222. Images of a single neuron using a confocal scanning microscope. The bottom image shows the merging of the green and red channels. B, same as in A but a group of neurons is shown. C, images of HEK293 cells after adeno-GFP infection and treatment with Ro31-8222. Note the 100 % co-localization of red and green fluorescence. Scale bars: 50 μm in A and C, 100 μm in B. D, percentage of cells expressing recombinant ρ1 receptors after adeno-ρ infection of HEK293 and 911 cell lines and hippocampal neurons over time. E, examples of GABA-activated currents on the second and fourth days after adeno-ρ infection of hippocampal cells. Note that on the fourth day, the GABA_A current was greatly diminished. Only a GABA_C-like current with the characteristic slow decay time was present.

Figure 4. Recombinant ρ1 receptors expressed in cultured hippocampal slices after adeno-ρ infection

A-C, images of cultured hippocampal slices after co-infection with adeno-GFP and adeno-ρ using a confocal scanning microscope. A, GFP fluorescence. B, staining with N-terminal ρ1 GABA_C receptor antibodies (red). C, co-localization of GFP and ρ1. Scale bar, 50 μm. D, example of a recombinant GABA_C current in the slice after adeno-ρ infection. The current was blocked by 3-APA, an antagonist of GABA_C receptors.