Inducible genetic suppression of neuronal excitability

Abstract

Graded, reversible suppression of neuronal excitability represents a logical goal of therapy for epilepsy and intractable pain. To achieve such suppression, we have developed the means to transfer "electrical silencing" genes into neurons with sensitive control of transgene expression. An ecdysone-inducible promoter drives the expression of inwardly rectifying potassium channels in polycistronic adenoviral vectors. Infection of superior cervical ganglion neurons did not affect normal electrical activity but suppressed excitability after the induction of gene expression. These experiments demonstrate the feasibility of controlled ion channel expression after somatic gene transfer into neurons and serve as the prototype for a novel generalizable approach to modulate excitability.

Fig. 1.

Ecdysone-inducible adenovirus constructs allow tight control of gene expression. A, Schematic representation of the basis for the ecdysone-inducible adenovirus vectors is shown. ψ, Packaging signal; Ecdpromoter, ecdysone-inducible promoter;IRES, internal ribosome entry site; ITR, inverted terminal repeat; MCS, multiple cloning site;pA, SV40 polyadenylation signal; RSV, Rous sarcoma virus; RXR, retinoid X receptor;VgEcR, modified ecdysone receptor.B, Infection of A549 cells with variable mois of both an adenovirus that contains an ecdysone-inducible promoter controlling expression of a cassette containing GFP, an IRES, and a multiple cloning site (AdEGI) and an adenovirus that expresses the modified ecdysone receptor and the retinoid X receptor (AdVgRXR) resulted in muristerone A–dependent and –inducible expression of GFP (▪ vs •). In the absence of AdVgRXR, background expression was minimal (▴), and such cells were entirely refractory to muristerone A (○). C, Expression of GFP in A549 cells, infected with both AdVgRXR and AdEGI, can be induced with 3 μmmuristerone A for the indicated amounts of time (columns 2–4), reduced to near background levels by the removal of muristerone A for 36 hr (column 5), and reinduced to the original induction level with muristerone A for 24 hr (column 6). D, Membrane currents from A549 cells infected with AdVgRXR and AdEGI-Kir2.1 in the absence (I = −5 pA/pF;n = 2) and presence (I = −80 ± 12 pA/pF; n = 4) of 3 μmmuristerone A are shown.

Fig. 2.

Infection of SCG neurons with inducible adenovirus constructs. A, B, Confocal images of SCG neurons infected with AdEGI-Kir2.1 and AdVgRXR in the absence (A) or presence (B) of muristerone A. C–E, A neuron expressing the fusion protein EGFP-Kir2.1 after fixation and staining with a polyclonal antibody to GFP and a monoclonal antibody against the MAP2. An FITC-labeled goat anti-rabbit secondary antibody was used to detect the presence of GFP (D), and a Cy3-conjugated secondary antibody was used to detect the bound anti-MAP2 (C). MAP2 is normally distributed in the dendrites and cell bodies.

Fig. 3.

Exogenous expression of Kir2.1 suppresses excitability. A, B, The effects of a suprathreshold stimulus on a control cell do not change with the addition of barium. C, The membrane current recorded from the same cell under these conditions using a ramp protocol from −138 to +22 mV over 500 msec is shown. The addition of barium had little or no detectable effect on the outwardly rectifyingI–V curves in control cells. D,E, The response to a subthreshold stimulus on a Kir2.1-infected cell (D) is markedly changed by the addition of barium (E); thearrow indicates the cessation of the applied stimulus.F, The I–V curve for this cell is much different after addition of barium. G–I, Membrane current recordings from a Kir2.1-infected cell are shown in normal saline (G), after superfusion with saline containing barium (H), and after washout (I).

Fig. 4.

Cumulative effects of exogenous expression of Kir2.1. A, Cells expressing Kir2.1 are hyperpolarized with respect to control cells. This effect is reversed after blocking the Kir2.1 channels. B, Increasing amounts of Kir2.1 current increase the size of the stimulus needed to fire an action potential.