Suppression of the rat microglia Kv1.3 current by src-family tyrosine kinases and oxygen/glucose deprivation.
Microglia activate following numerous acute insults to the brain, including oxygen/glucose deprivation (OGD), and both protein tyrosine kinases (PTKs) and K+ channels have been implicated in their activation. We identified Kv1.3 (voltage-gated potassium channel) protein in cultured rat microglia and confirmed that the native current is biophysically and pharmacologically similar to Kv1. 3. To explore whether src-family PTKs regulate the microglial Kv current, we first heterologously expressed Kv1.3 in a microglia-like cell line derived from neonatal rat brain (MLS-9). The resulting large Kv1.3 current was eliminated by co-transfecting the constitutively active PTK, v-src, then rapidly restored by the PTK inhibitor, lavendustin A. Acute activation of endogenous src kinases by a peptide activator significantly reduced the current, an effect that was mimicked by OGD. Similarly, in primary cultures of rat microglia, the endogenous Kv1.3-like current was inhibited by activating endogenous src-family PTKs and by OGD. Biochemical analysis showed that OGD increased the tyrosine phosphorylation of native Kv1.3 protein, which was alleviated by PTK inhibitors or reactive oxygen species (ROS) scavengers. Conversely, the basal level of Kv1.3 phosphorylation was decreased by PTK inhibitors or scavengers of ROS. Together, our results point to a post-insertional downregulation of the microglial Kv1.3-like current by oxidative stress and tyrosine phosphorylation. This interaction may be facilitated by a multiprotein complex because, in cultured microglia, the endogenous Kv1.3 and src proteins both bind to the scaffolding protein, post-synaptic density protein 95 (PSD-95). By associating with, and phosphorylating Kv1.3, src is well positioned to regulate microglial responses to oxidative stress.
Cayabyab
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Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Canada.
Khanna
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Jones
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Schlichter
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D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D000076122:Disks Large Homolog 4 Protein; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D051664:Kv1.3 Potassium Channel; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009419:Nerve Tissue Proteins / Q000235:genetics; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D010100:Oxygen / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D017208:Rats, Wistar; D012604:Scorpion Venoms / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; C496975:Kcna3 protein, rat; D051664:Kv1.3 Potassium Channel; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; C033652:postsynaptic density proteins; C087603:agitoxin 2; D014443:Tyrosine; D015688:Oncogene Protein pp60(v-src); D019061:src-Family Kinases; D005947:Glucose; D010100:Oxygen