Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3

Abstract

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca(2+)](i)), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca(2+)](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 Ca(2+) imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca(2+)](i) and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca(2+)-dependent, TRAM-34-sensitive K(Ca)3.1 channels, and Ca(2+)-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 Ca(2+)/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 Ca(2+)-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 [Ca(2+)](i) increases, which likely play similar roles in other migratory cells in the nervous system.

Figure 1.

Bradykinin induces [Ca²⁺]_i elevations that correlate with electrophysiological changes in human glioma cells. A–C, D54 human glioma cells. D–F, U87 human glioma cells. A, D, At time = 0 s, fura-2 340/380 ratio is depicted in the top. Bottom trace depicts current–voltage curve for the same cell as measured with a perforated-patch pipette. B, E, After application of 10 μm bradykinin (BK), there is a large increase in [Ca²⁺]_i (top) and a leftward shift of the reversal potential (bottom). C, F, After [Ca²⁺]_i has returned to basal levels (top), there is a large rightward shift in the reversal potential (bottom).

Figure 2.

Bradykinin-induced [Ca²⁺]_i elevations activate K_Ca3.1 channels and Cl⁻ channels in human glioma cells. A, Fura-2 340/380 ratios after application of 10 μm bradykinin (BK). F/F₀ indicates fluorescence normalized to fluorescence at t = 0 s. B, Percentage of cells responding to BK with a rise in [Ca²⁺]_i. C, D, Time to BK-induced peak hyperpolarization and depolarization, respectively. E–H, Representative cells for BK, BK plus TRAM-34, BK plus gluconate (gluc), and BK plus BAPTA-AM conditions. Top traces depict [Ca²⁺]_i versus time. For the same cell at the same time points, bottom traces depict membrane potential (V_m) at I = 0 pA versus time. I, Peak early-phase voltage change (millivolts). J, K, BK-induced current–voltage changes during early-phase response. L, Peak late-phase voltage change (millivolts). M, N, BK-induced current–voltage changes during late-phase response. n = 10–21 cells; *p < 0.05.

Figure 3.

Increases in [Ca²⁺]_i activate TRAM-34-sensitive K_Ca3.1 channels and DIDS- and gluconate⁻-sensitive Cl⁻ channels. A, Representative TRAM-34-sensitive K_Ca3.1 currents elicited after a step protocol from −100 to 120 mV at each of the [Ca²⁺]_i listed. B, TRAM-34-sensitive current–voltage relationship at 0, 65, and 180 nm [Ca²⁺]_i. C, TRAM-34-sensitive K_Ca3.1 current density at −40 mV. n = 5–8 cells. D, Representative DIDS-sensitive Cl⁻ currents elicited after a step protocol from −100 mV to 120 mV at each of the [Ca²⁺]_i listed, with or without extracellular gluconate⁻ replacement. E, DIDS-sensitive current–voltage relationship at 0, 65, and 180 nm [Ca²⁺]_i, with or without extracellular gluconate⁻ replacement. F, DIDS-sensitive Cl⁻ current density at 40 mV. n = 5–10 cells; *p < 0.05. gluc, Gluconate.

Figure 4.

ClC-3, K_Ca3.1, and B₂R are expressed on the leading edges of human glioma cells. A, Field of view of cultured D54 human glioma cells labeled with antibodies targeted against ClC-3 (blue), K_Ca3.1 (green), and B₂R (red). Zooms of boxed cells depicted in B and C. Scale bar, 20 μm. B, C, Zooms of individual cells depicted in A. Scale bar, 10 μm. n = 3. D, Several human glioma lines express ClC-3, K_Ca3.1, and B₂R protein. n = 3.

Figure 5.

A subset of Ca²⁺-activated Cl⁻ currents are mediated by CaMKII-dependent ClC-3 channels. A, Representative DIDS-sensitive Cl⁻ currents elicited after a step protocol from −100 to 120 mV at each of the [Ca²⁺]_i listed, with or without AIP preincubation to inhibit CaMKII. B, DIDS-sensitive current–voltage relationship at 0, 65, and 180 nm [Ca²⁺]_i, with or without AIP. C, DIDS-sensitive Cl⁻ current density at 40 mV. n = 10 cells. D, Representative Western blot demonstrating knockdown of ClC-3 expression. GAPDH used as a loading control. E, Representative DIDS-sensitive Cl⁻ currents elicited after a step protocol from −100 to 120 mV at each of the [Ca²⁺]_i listed, with NT or ClC-3 shRNA transfected cells. F, DIDS-sensitive current–voltage relationship at 0, 65, and 180 nm [Ca²⁺]_i, with or without ClC-3 expression. G, DIDS-sensitive current–voltage relationship at 65 and 180 nm [Ca²⁺]_i, with simultaneous ClC-3 knockdown and CaMKII inhibition with AIP. H, DIDS-sensitive Cl⁻ current density at 40 mV. n = 5–13 cells; *p < 0.05.

Figure 6.

Bradykinin-induced Cl⁻ currents are partially mediated by CaMKII activation of ClC-3. A, Time to BK-induced peak depolarization after inhibition of CaMKII with AIP and knockdown of ClC-3. B, Peak late-phase voltage change (millivolts) after inhibition of CaMKII with AIP and knockdown of ClC-3. C, D, BK-induced current–voltage changes during late-phase response. CaMKII or ClC-3 inhibition decreases bradykinin-induced Cl⁻ currents. n = 15 cells; *p < 0.05.

Figure 7.

Bradykinin-induced glioma cell migration requires K_Ca3.1 channel and CaMKII-dependent ClC-3 channel activity. A, Diagram depicting glioma cells plated onto a Transwell barrier containing 8 μm pores. Bradykinin (BK) loaded on opposite side of filter to assess chemotactic migration. B, Normalized number of glioma cells migrated under various conditions. Ion channel inhibition eliminates bradykinin-induced migration. n = 4; *p < 0.05.

Figure 8.

Bradykinin-induced glioma cell invasion through cerebral parenchyma requires ion channel activity. A, Representative mouse brain slices containing large bradykinin (BK)-induced EGFP-labeled human glioma tumor, which is inhibited by TRAM-34, icatibant, and ClC-3 knockdown. B, C, Quantification demonstrates that BK-induced tumor spreading and growth is suppressed by ion channel or B₂R inhibition. n = 3–14; *p < 0.05. D, Human tissue lysates probed for ClC-3, K_Ca3.1, and B₂R. NB, Normal brain; IV, grade IV glioblastoma. n = 3. E, BK binds to GPCR (1) and leads to increases in [Ca²⁺]_i (2). These increases in [Ca²⁺]_i activate K_Ca3.1 channels and K⁺ efflux (3). Increases in [Ca²⁺]_i also activate Cl⁻ channels, including CaMKII activation of ClC-3, leading to Cl⁻ efflux (4). K⁺ and Cl⁻ efflux lead to osmotic loss of cytosolic water. A loss in cytosolic ions and water enables cellular volume and shape changes, facilitating glioma cell migration through narrow extracellular spaces (5).