The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

Abstract

BackgroundSmall-conductance Ca2+ activated K+ channels are expressed in the CNS, where KCNN2/SK2/KCa2.2 and KCNN3/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of KCNN3/SK3 in CNS microglia in vivo and in vitro, and to use an established in vitro model to determine if this channel contributes to the neurotoxic capacity of activated microglia.MethodsKCNN3 mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-κB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke).ResultsKCNN3 mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells.ConclusionsSK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic in vitro studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.

Swelling activated Cl- channels in microglia

“…We found that some ion channels in microglia contribute to their neurotoxic capacity, and identified roles of three K + channel types (K v 1.3, K Ca 2.3, K Ca 3.1) in production of nitric oxide, superoxide and peroxynitrite. [7][8][9] After acute CNS injury (e.g., stroke, trauma), the excitatory amino acid, glutamate, causes neuronal injury; thus, a crucial finding was that I Clswell channels in microglia are significantly permeable to glutamate (and . When the bath was perfused with the hypo-osmotic solution 5, the cell swelled (inset) and an outward-rectifying current activated, increased with time (vertical arrow) and reached a quasi-stationary plateau.…”

“…Furthermore, after injury, microglia migrate to the site of injury, as shown in our recent studies of K + channels in rat models of optic nerve transection, ischemic stroke and intracerebral hemorrhage. [8][9][10] In addition, glutamate is involved in signaling between neurons, astrocytes and microglia; thus, potential outcomes of activating I Clswell include trans-activating NMDA receptors to evoke excitotoxic neuronal injury; 11 activating microglia through their metabotropic glutamate receptors, leading to production of other neurotoxic mediators; 12,13 and mediating glia-to-neuron signaling under inflammatory conditions. 14 Here, we first show that the biophysical and pharmacological properties of I Clswell in MLS-9 cells are as previously determined for primary rat microglia.…”

Physiology of Microglia

“…The messages for KCNN1/SK1, KCNN2/SK2, and KCNN3/SK3 channels were identified in rat cultured microglia; the KCNN3/SK3 showed predominant expression, which increased ϳ1.7 times following LPS stimulation (807). The immunoreactivity for KCNN3/ SK3 was found in microglial cells in healthy adult rat striatum; this immunoreactivity substantially increased after ischemic lesion (807). Finally, inhibition of KCNN3/ SK3 channels by 100 nM apamin or by 5 nM tamapin reduced microglial neurotoxicity and somewhat ameliorated microglial activation (807).…”

“…The K Ca channels are also involved in controlling the microglial respiratory burst; the treatment of microglial cells with apamin (selective blocker of KCNN2/SK2 channels) or with clotrimazole (KCNN4/SK4 channels inhibitors) markedly inhibited the respiratory burst (446). The messages for KCNN1/SK1, KCNN2/SK2, and KCNN3/SK3 channels were identified in rat cultured microglia; the KCNN3/SK3 showed predominant expression, which increased ϳ1.7 times following LPS stimulation (807). The immunoreactivity for KCNN3/ SK3 was found in microglial cells in healthy adult rat striatum; this immunoreactivity substantially increased after ischemic lesion (807).…”

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

“…In particular, it produces enhanced pain transmission in the spinal dorsal horn after spinal cord injury (Gwak & Hulsebosch, 2011). Recently, the SK3 ion channel has gained attention as a new therapeutic target for disorders involving neuron hyper-excitability (Schlichter et al, 2010). Results from the present study suggest direct interactions between Na-ASP-2 and extracellular SK3 peptides, thus raising the hope that this hookworm protein may hold promise as a new SK3 channel modulator with therapeutic potential.…”