Bradykinin-Induced Chemotaxis of Human Gliomas Requires the Activation of KCa3.1 and ClC-3

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca2+](i)), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of humangliomas to study how[Ca2+](i) regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca2+ imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca2+](i) and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca2+-dependent, TRAM-34-sensitive K(Ca)3.1 channels, and Ca2+-dependent, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl- channels. A significant portion of these Cl- currents can be attributed to Ca2+/calmodulin dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl- channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca2+-activated Cl- currents. Western blots show that K(Ca)3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both K(Ca)3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slices in situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca2+](i) increases, which likely play similar roles in other migratory cells in the nervous system.