Guanosine promotes the up‐regulation of inward rectifier potassium current mediated by Kir4.1 in cultured rat cortical astrocytes

Abstract

Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long-term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 lM) promotes the functional expression of an inward rectifier K + (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up-regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo-induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo-induced up-regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that downregulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K + homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K + -buffering impairment of the astroglial syncytium that occurs in pathological conditions.

Molecular Mechanisms of EAST/SeSAME Syndrome Mutations in Kir4.1 (KCNJ10)

“…The biophysical properties of EGFP-tagged wildtype channels (pH dependence and rectification; see Figs. 4 and 5) were identical to those reported for nontagged channels in native cells (37) and heterologous expression systems (5,6). However, we cannot discount the possibility that the fluorescent tag might affect localization or trafficking.…”

Guanosine and its role in neuropathologies

“…In agreement, these signaling pathways have been associated with the regulation of astrocytic glutamate transporters [142][143][144]. Furthermore, in vitro studies in cultured astrocytes have also suggested that guanosine exerts its effects by increasing the expression of inward rectifying K + (Kir) channels [145]. The involvement of K + channels was further supported by the observation that the protective effects of this nucleoside against OGD followed by reoxygenation in hippocampal slices are dependent on the activation of Ca 2+ -activated K + (BK) channels [140].…”

An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes

“…Primary cultured rat cortical astrocytes were prepared, and DI TNC1 cells were cultured and maintained as described previously (31). DI TNC1 cells were transfected with the pEGFP-N1 construct or cotransfected with pEGFP-N1 + AQP4-M1 or + AQP4-M23 by use of Lipofectamine 2000, following the manufacturer's instructions (Invitrogen).…”

“…Biotinylation of cell-surface membrane proteins was performed on cultured astrocytes as previously described (31). To test the specificity of the assay, the same experiment was performed without the addition of biotin donor.…”