Figure 4.

SMN-deficient spinal astrocytes show a reduction in K_ir4.1 protein level and modulation in electrophysiological properties. (A) Immunostaining of wild-type astrocyte cultures transfected with scrambled (scr.) or SMN siRNA against K_ir4.1 protein (green) at DIV 10. Nuclear DNA was stained with Dapi (blue). (B) SMN-deficient spinal astrocytes showed reduced protein levels of K_ir4.1, compared to astrocytes transfected with scrambled siRNA (***, p < 0.001). (C) Raw trace of current–voltage (IV) curve of K_ir4.1 channel protein in cultured spinal astrocytes transfected with scrambled or SMN siRNA or scrambled siRNA + K_ir4.1 inhibitor VU. SMN-deficient astrocytes showed reduced current as a sign of reduced potassium uptake into spinal astrocytes. A similar effect was observed when VU inhibited the Kir4.1 function. (D) Current density of K_ir4.1 was reduced in SMN-deficient spinal astrocytes, compared to astrocytes transfected with scrambled siRNA (***, p < 0.001). When K_ir4.1 function was inhibited in wild-type astrocytes exposed to scrambled siRNA, the current density was reduced (***, p < 0.001). (E) Astrocytes transfected with SMN siRNA showed depolarized resting membrane potential (***, p < 0.001). A similar result was observed when K_ir4.1 function was inhibited by VU (***, p < 0.001). n = 3 independent experiments for immunostaining (see bars). For each experiment > 50 cells were analyzed. n = 7 cells per electrophysiological measurement (see bars). Scale bar: 50 µm.