Expression, roles and regulation of potassium channels in neuroimmune cells.

摘要

CNS diseases and damage result in infiltration of immune cells and activation of brain-resident microglia. We are interested in the molecular expression, regulation and roles of selected potassium channels in human T lymphocytes and rat brain microglia, as possible therapeutic targets for controlling brain inflammation. Our results point to roles for specific potassium channels.; We found that a Ca2+-activated K+ channel that regulates Ca2+-signaling events during T-cell activation is encoded by the SK4 gene. SK4 mRNA and current are up-regulated after mitogenic stimulation, as is its role in proliferation and regulatory volume decrease. SK4's exquisite Ca2+ sensitivity is conferred by calmodulin binding to the proximal carboxyl-terminal domain, which we call “Ct1”. Moreover, assembly and localization appear to require calmodulin-dependent linking of SK4 monomers by Ct1, since: (i) CaM increased cell-surface SK4 without changing its expression level; (ii) Over-expression of the calmodulin-binding Ct1, but not other regions, reduced the wild-type current—an effect abrogated by calmodulin over-expression; (iii) SK4 multimerization was increased by CaM over-expression and decreased by Ct1 over-expression.; Since microglia activation often includes proliferation and an NADPH oxidase-mediated respiratory burst, we examined the roles of specific K + channels in these processes. Cultured rat microglia expressed voltage-gated Kv1.3 and Kv1.5 channels, and Ca2+ (and calmodulin)-gated SK2, SK3 and SK4 channels at the mRNA level. Kv1.3, Kv1.5 and SK3 proteins were detected, and the three distinct K+ currents biophysically and pharmacologically resembled Kv1.3, SK2/SK3 and SK4. All three currents contributed significantly to the respiratory burst, but not to proliferation in these highly purified microglial cultures. Kv1.3 is subject to post-insertional inhibition, since it was dramatically reduced by activation of the src protein tyrosine kinase (PTK), but rapidly restored by the PTK inhibitor, lavendustin A. Oxygen-glucose deprivation—an in vitro model of stroke—mimicked Kv1.3 inhibition and increased its tyrosine phosphorylation, an effect alleviated by PTK inhibitors or scavengers of reactive oxygen species (ROS). This modulation may be facilitated by a multi-protein complex since, in microglia, Kv1.3 and src bind to the scaffolding protein, PSD-95. By associating with, and phosphorylating Kv1.3, src is well positioned to regulate microglial responses to oxidative stress.