Bradykinin-Induced Chemotaxis of Human Gliomas Requires the Activation of K_Ca3.1 and ClC-3

Abstract

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 largely unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca2+]i regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers which spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin activating G protein-coupled receptors. 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 KCa3.1 channels, and Ca2+-depdenent, DIDS- 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, since pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca2+-activated Cl− currents. Western blots show that KCa3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with Grade IV gliomas. Both KCa3.1 and ClC-3 co-localize 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, this data elucidates 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.

Molecular Mechanisms of Glioma Cell Motility

“…The KCa family of Ca 2 + -activated K + channels, especially KCa3.1, is overexpressed in 32% of the glioma patients, and its expression correlates with patient survival (126). KCa3.1 is localized at the leading edge of migrating cells, and its inhibition results in reduced migration (127,128). The bradykinin receptor B 2 (B 2 R) is also expressed at the leading edge of migrating glioma cells.…”

“…The bradykinin receptor B 2 (B 2 R) is also expressed at the leading edge of migrating glioma cells. It is a critical attractor of glioma cells toward the vasculature, and an activator of ion channels (127,129). Binding of bradykinin to B 2 R leads to increases in intracellular Ca 2 + which induces the opening of the KCa3.1 and ClC-3 channels, resulting in the efflux of Cl − , K + , and water (16,127,130).…”

The Roles of Cycloxygenase and Endothelial Derived Hyperpolarizing Factors in Bradykinin-Induced Aortic Relaxation

“…This has been explained on the bases of BK activates K + efflux through KCa2+ involving both BKCa and SKCa (Liu, Freyer, & Hall, 2007), and such activation are suppressed by TEA preincubation. Recently, (Cuddapah, Turner, Seifert, & Sontheimer, 2013) demonstrated that KCa2+ channels are activated as a result of BK-dependent Ca 2+ increase.The main reason behind the vasoconstriction effect of TEA is that the muscle is under depolarization and Ca +2 enter the cells while BKCa +2 channel opens during hyperpolarization and cause relaxation (Sanchez & López-Zapata, 2011).…”

Cl − and K + channels and their role in primary brain tumour biology

“…[38,39]. Pharmacological inhibition or genetic knockdown of KCa3.1 disrupts chemotaxis towards either of these ligands [38,39].…”

“…[38,39]. Pharmacological inhibition or genetic knockdown of KCa3.1 disrupts chemotaxis towards either of these ligands [38,39]. KCa3.1 is also required for the migration of neuroblasts, one of the purported brain tumour precursor cells, along the RMS [40].…”