Elsevier

Cell Calcium

Volume 53, Issue 3, March 2013, Pages 187-194
Cell Calcium

Calcium entry via TRPC1 channels activates chloride currents in human glioma cells

https://doi.org/10.1016/j.ceca.2012.11.013Get rights and content

Abstract

Malignant gliomas are highly invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl channels are regulated in the context of migration. Using patch-clamp recordings we show Cl currents are activated by physiological increases of [Ca2+]i to 65 and 180 nM. Cl currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl currents in a Ca2+-dependent fashion, placing Cl channels under the regulation of Ca2+ entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl channels. Thus endogenous glioma Cl channels are regulated by TRPC1. Cl channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca2+]i to the shape and volume changes associated with migrating cells.

Introduction

Malignant gliomas are the most common and deadly form of primary brain cancer affecting adults. These cancers are typified by an elevated mitotic index and robust invasiveness. The invasiveness is facilitated by ion channels, which allow cellular shape and volume changes during migration, allowing cells to squeeze through narrow extracellular spaces. Ion channels control cellular shape and volume by moving ions, which are osmolytes, through the plasma membrane, osmotically drawing water across the cell membrane [1]. Thus, ion channels, by allowing hydrodynamic volume changes, facilitate cell migration. In glioma cells, several of the ion channels responsible for these processes have been identified and include Ca2+-activated K+ channels [2] and voltage-activated Cl channels/transporters [3].

Members of the ClC family of voltage-activated Cl channels/transporters endogenously expressed by glioma cells include ClC-2, -3, and -5 [4]. Of these channels, ClC-3 is expressed on the plasma membrane, facilitates volume and shape changes, and promotes glioma migration [3], [5]. While ClC-3 seems to play an important role in migration, little is known about how ClC-3 becomes activated. We recently demonstrated that ClC-3 can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells [3] thereby coupling changes in [Ca2+]i to changes in Cl channel activity. Upon phosphorylation, ClC-3 currents increase 3-fold, and inhibition of ClC-3 phosphorylation significantly reduces glioma cell migration [3]. Given that glioma Cl channels are regulated by a Ca2+-sensitive kinase, the goals of the present study were to (1) determine if elevations in [Ca2+]i are sufficient to increase Cl conductance, and (2) identify a physiological source for this Ca2+.

A potential source for Ca2+ in glioma cells includes transient receptor potential canonical (TRPC) channels, non-selective cation channels permeable to Na+, K+, Mg2+, and Ca2+. Gliomas express TRPC-1, -3, and -5, which give rise to currents blocked by GdCl3, 2-APB, or SKF96365 [6]. TRPC1 plays a role in store-operated Ca2+ entry [7] and regulates EGF-induced chemotaxis in glioma cells [8]. Hence we ask whether TRPC1 is a source for Ca2+ to activate Cl channels in glioma cells.

Using inducibly expressed TRPC1 shRNA, we find that Ca2+ entry through TRPC1 activates glioma Cl channels. We show that Cl channels are regulated by physiologically relevant changes in [Ca2+]i, i.e. between 0–180 nM. Such [Ca2+]i changes occur in response to TRPC1 activity, e.g. during EGF-stimulated chemotaxis. Consistent with this we find prominent colocalization of ClC-3 and TRPC1 in caveolar lipid rafts on the processes of glioma cells. This interaction appears to be functionally relevant during EGF-induced chemotaxis.

Section snippets

Cell culture

D54 human glioma cells were derived from a World Health Organization grade IV glioblastoma and gifted to us by Dr. D. Bigner (Duke University, Durham, NC). D54 cells were maintained in Dulbecco's modified Eagle's medium/F-12 (DMEM-F/12), supplemented with 2 mM glutamine and 7% fetal bovine serum (Hyclone, Logan, UT). Cells were incubated in a humidified chamber at 37 °C and 10% CO2. Reagents were purchased from Sigma-Aldrich unless otherwise noted.

Electrophysiology

Whole-cell patch clamp recordings were performed

[Ca2+]i regulates Cl currents in human glioma cells

Previous studies demonstrate that human glioma cells express voltage-gated Cl channels, which modulate cellular volume to promote cell motility [1], [3], [10]. Given that a subset of these Cl channels are regulated by Ca2+-sensitive kinases [3], [11], we hypothesize that [Ca2+]i may also regulate Cl channel activity. To test this hypothesis, we first performed whole-cell patch clamp experiments on human glioma cells. By manipulating the [Ca2+] of the pipette solution, we changed [Ca2+]i in

Discussion

To our knowledge, this is the first demonstration that increases in [Ca2+]i leads to enhanced Cl channel activity in gliomas (Fig. 1). As previously reported, Cl channels play an important role in the shape and volume changes associated with migrating cells [1], [19]. This led us to ask how Cl channels are physiologically regulated in human glioma cells. We found that knockdown of TRPC1 decreased Cl conductance in a Ca2+-dependent manner. These currents were likely mediated by ClC-3, a

Acknowledgments

We thank Dr. Valerie Bomben for creating the inducible cell lines and Dr. Susan L. Campbell for critical reading of the manuscript. We are grateful for the following grants from the National Institutes of Health (National Institute of Neurological Disorders and Stroke): RO1 NS036692 and RO1 NS031234 to H.S. and F31 NS073181 to V.A.C.

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