Abstracts

Tuberous sclerosis complex (TSC) is a genetic disease characterized by severe epilepsy and neuropathological abnormalities involving astrocytes. Astrocyte-specific inactivation of the [italic]Tsc1[/italic] gene in mice results in seizures and premature death. We have hypothesized that epileptogenesis due to a primary astrocytic defect in [italic]Tsc1[/italic] conditional knock-out ([italic]Tsc1[/italic] cKO) mice may be related to impairment of uptake of excitatory substances in the neuronal extracellular environment. We previously demonstrated a reduction in glutamate transporter activity in [italic]Tsc1[/italic] cKO astrocytes. In the present study, we examined the hypothesis that impairment of astrocytic K⁺uptake through inward-rectifying K⁺ channels may also represent a mechanism of epileptogenesis in [italic]Tsc1[/italic] cKO mice.
Astrocyte cultures were established from postnatal day 2 control and [italic]Tsc1 [/italic]cKO mice. Whole-cell recordings were obtained using patch pipettes from isolated astrocytes maintained in culture for 7 to 12 days. Membrane currents were elicited by voltage steps from a holding potential of -70 mV. Current through inward-rectifying K⁺ channels was isolated by subtraction of signals obtained in the absence and presence of 100 [mu]M barium, which selectively blocks these channels. Potassium conductance was calculated by linear regression analysis of the hyperpolarized portion of the current-voltage plot. Western blot analysis of inward-rectifying K⁺ channel subtypes was performed using standard techniques.
In the absence of barium, the resting membrane potential was slightly, although significantly, depolarized in [italic]Tsc1[/italic] cKO astrocytes compared to controls. Hyperpolarizing and depolarizing voltage steps from the holding potential of -70 mV elicited large current responses, with significantly lower conductance values in [italic]Tsc1[/italic] cKO astrocytes than in control astrocytes (540 vs. 880 pS/pF, p[lt]0.05). Application of 100 [mu]M barium to block inward-rectifying K⁺ channels caused a significant depolarizing shift in resting membrane potential and inhibited a large portion (45-100%) of the total current. Analysis of the barium-sensitive portion of the current revealed a weak inward rectification. Conductance through these inward-rectifying channels was significantly lower in [italic]Tsc1[/italic] cKO astrocytes than in controls (380 vs. 770 pS/pF, p[lt]0.05). Western blot analysis of inward-rectifying K⁺ channel subtypes demonstrated differential expression in [italic]Tsc1[/italic] cKO and control astrocytes.
Inward-rectifying K⁺ channel currents are reduced in [italic]Tsc1[/italic] cKO astrocytes. This impairment may result in abnormal extracellular potassium homeostasis, which, in concert with the previously demonstrated reduction in glutamate transporter activity, may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.
[Supported by: Tuberous Sclerosis Alliance (DHG), NIH 5K12NS0169004 (MW) ]