Potassium channel activity and glutamate uptake are impaired in astrocytes of seizure-susceptible DBA/2 mice

Abstract

Purpose: KCNJ10 encodes subunits of inward rectifying potassium (Kir) channel Kir4.1 found predominantly in glial cells within the brain. Genetic inactivation of these channels in glia impairs extracellular K(+) and glutamate clearance and produces a seizure phenotype. In both mice and humans, polymorphisms and mutations in the KCNJ10 gene have been associated with seizure susceptibility. The purpose of the present study was to determine whether there are differences in Kir channel activity and potassium- and glutamate-buffering capabilities between astrocytes from seizure resistant C57BL/6 (B6) and seizure susceptible DBA/2 (D2) mice that are consistent with an altered K(+) channel activity as a result of genetic polymorphism of KCNJ10.

Methods: Using cultured astrocytes and hippocampal brain slices together with whole-cell patch-clamp, we determined the electrophysiologic properties, particularly K(+) conductances, of B6 and D2 mouse astrocytes. Using a colorimetric assay, we determined glutamate clearance capacity by B6 and D2 astrocytes.

Results: Barium-sensitive Kir currents elicited from B6 astrocytes are substantially larger than those elicited from D2 astrocytes. In addition, potassium and glutamate buffering by D2 cortical astrocytes is impaired, relative to buffering by B6 astrocytes.

Discussion: In summary, the activity of Kir4.1 channels differs between seizure-susceptible D2 and seizure-resistant B6 mice. Reduced activity of Kir4.1 channels in astrocytes of D2 mice is associated with deficits in potassium and glutamate buffering. These deficits may, in part, explain the relatively low seizure threshold of D2 mice.

Fig. 1

A and C: I–V curves from astrocytes of B6 and D2 mice. Whole-cell current-voltage relations of astrocytes from B6 mice (A; n=10) and D2 mice (C; n=19) in the absence (circles) and presence (inverted triangles) of 200 μM barium. Currents were recorded in response to 20 mV steps from −100 mV to +100 mV from Vm. In these experiments, the cell was held at the steady state potential (Vh=Vm). The mean Vm values and S.E.M. are −51.5 ± 4.1 and −45.9 ± 3.2 for B6 and D2 astrocytes, respectively. These values are not statistically different. B and D: Representative current traces from astrocytes of B6 and D2 mice in response to 20 mV steps from 100 mV to +100 mV from Vm. B shows representative current traces from a B6 astrocyte in the absence (B1) and presence (B2) of 200 μM barium. D shows representative current traces from a D2 astrocyte in the absence (D1) and presence (D2) of 200 μM barium.

Fig. 2

Summary of the current responses of B6 and D2 astrocytes to changes in [K⁺]_o. The astrocytes were bathed in solution containing 3 mM K⁺ and this solution was changed to 1, 10 or 30 mM K⁺ and the current response measured using the whole-cell voltage clamp technique. (n=12 and 13 for B6 and D2, respectively). * indicates significant difference from B6 group (p<0.01; Student’s t-Test for independent samples). In these experiments, the cell was held at the steady state potential (Vh=Vm). The mean Vm values and S.E.M. are −55.8 ± 3.3 and −48.8 ± 4.6 for B6 and D2 astrocytes, respectively. These values are not statistically different.

Fig. 3

A. Representative whole cell currents recorded from B6 and D2 astrocytes in hippocampal brain slices. Inward currents were obtained by changing extracellular K⁺ from 2.5 mM to 10 mM in the presence or absence of 100 μM Ba²⁺. The cells were held at the steady state potential (Vh=Vm). The scales bars are equal for all current traces in Part A. B. Summary of the relative barium sensitive Kir currents measured in B6 and D2 astrocytes (n= 9 and 14, respectively). The data are expressed as % of control current that is barium sensitive where control is the maximal current measured by switching extracellular [K+] from 2.5 to 10 mM. * indicates significant difference from B6 group (p<0.01; Student’s t-Test for independent samples). The mean Vm values and S.E.M. are −82.6 ± 1.1 and −85.6 ± 2.2 for B6 and D2 astrocytes, respectively. These values are not statistically different.

Fig. 4

The effect of TBOA on glutamate clearance by B6 and D2 astrocytes. The concentration of glutamate was determined using a colorimetric assay 60 min after addition of 400 μM glutamate in medium and compared with the concentration of glutamate measured in the absence of astrocytes. B6 astrocytes cleared significantly more glutamate in 60 mins than D2 astrocytes. TBOA (200μM) completely blocked glutamate clearance by both B6 and D2 astrocytes. * indicates significant difference from control (no astrocytes) and # indicates a significant difference between the B6 and D2 astrocytes (p<0.01; ANOVA followed by Tukey’s test) with n = 12 per group.