The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke

Abstract

Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K þ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K þ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K þ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/ macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1 À/À mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke

“…Animals that met these inclusion criteria were assigned to treatment or vehicle groups based on a computer‐generated randomization scheme. Based on our previous work with the KCa3.1 blocker TRAM‐34 in MCAO,16, 25 sample size was powered to detect a reduction of 40% in mean percentage infarct area with 80% power. Starting 12 h after reperfusion animals received the vehicle miglyol 812 neutral oil (Neobee M5 ® , Spectrum Chemicals) or PAP‐1 at 10 or 40 mg/kg intraperitoneally every 12 h until sacrifice on day‐8.…”

“…CD11b + cells from the infarct area were isolated as previously described 16, 27. Briefly, mice were anesthetized with isoflurane 2, 5, and 8 days after reperfusion MCAO and killed by cervical dislocation.…”

“…NeuN (1:1000, A60, Millipore) staining to assess infarct area was performed as described 16. For the mouse brain IF images in Figure 1A, Kv1.3 was stained with a polyclonal anti‐Kv1.3 antibody (1:300, APC‐101, Alomone) and Iba1 with a monoclonal anti‐Iba1 antibody (1:1000, NCNP24, Wako Chemical).…”

“…Electrophysiological experiments on slices15 or acutely isolated microglia16 have further demonstrated Kv1.3 currents on activated microglia following MCAO. In humans, strong Kv1.3 staining has been reported on microglia in the frontal cortex of patients with Alzheimer's disease17 and on CD68 + cells in multiple sclerosis brain lesions 18.…”

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore

“…Similar to the related small-conductance Ca 21 -activated K 1 channel or KCa2 channels, KCa3.1 channels have fewer charges in their S4 segment than voltage-gated K 1 (Kv) channels (Wei et al, 2005); therefore, they do not respond to changes in membrane voltage but instead are activated by Ca 21 binding to calmodulin, which functions as their b-subunit and induces Ca 21 -dependent channel opening (Fanger et al, 1999 KCa3.1 is widely expressed in cells of the immune system such as T-and B-lymphocytes (Ghanshani et al, 2000;Wulff et al, 2004), mast cells (Shumilina et al, 2008), macrophages, and microglia (Chen et al, 2016), where it plays an important role in cellular activation, migration, and cytokine production (Feske et al, 2015). KCa3.1 is further important for volume regulation in erythrocytes (Vandorpe et al, 1998), which is why the channel is often referred to as the Gárdos channel after the Hungarian scientist, who first described a Ca 21 -activated K 1 efflux leading to erythrocyte dehydration (Gardos, 1958).…”