Mislocalization of K_ir channels in malignant glia

Abstract

Inwardly rectifying potassium (K ir ) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (E K ) and are implicated in potassium buffering. A number of previous studies suggest that K ir channel expression is indicative of cell differentiation. We therefore set out to examine K ir channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG-1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K + currents, depolarized resting membrane potentials (V m )(-38.5 ± 4.2 mV, D54 and -28.1 ± 3.5 mV, STTG1), and relatively high input resistances (R m ) (260.6 ± 64.7 MΩ, D54 and 687.2 ± 160.3 MΩ, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K + currents, had a mean V m of -51.1 ± 3.7 and a mean R m value of 627.5 ± 164 MΩ. In contrast, mature astrocytes had a significantly more negative resting membrane potential (-75.2 ± 0.56 mV), and a mean R m of 25.4 ± 7.4 MΩ. Barium (Ba 2+ ) sensitive K ir currents were >20-fold larger in mature astrocytes (4.06 ± 1.1 nS/pF) than in glioma cells (0.169 ± 0.033 nS/pF D54, 0.244 ± 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 ± 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several K ir channel subunits in glioma cells (K ir 2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (K ir 2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of K ir channel proteins to intracellular compartments is responsible for a lack of appreciable K ir currents in glioma cells.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

“…In fact, in GBMs, Kir 2.1 was detected in 95% of the cases, whereas a detectable I IRK was only found in 57% of the GBM tested, with a current density significantly lower than in the controls (Table 2). Our results are consistent with those reported by Olsen and Sontheimer (2004), who found a reduction of I IRK in glioma cell lines, due to diminished translation and mislocalisation of the Kir 2.1 protein. Our data suggest that such a diminished translation (or mislocalisation) of IRK-type channels occurs not only in high-grade glioma cell lines but also in tumour-derived GBM cells.…”

“…Bottom panels: by subtracting the currents in the presence of WAY from the control currents, we isolated hERG components, which turned out to be negligible in healthy tissue. Olsen and Sontheimer, 2004). As described in the Introduction, we sought IRK-type inwardly rectifying currents (I IRK ), and hERG (I hERG ), or hELK2 (I hELK2 ) currents.…”

“…In addition, RT -PCR hERG1 channels and glioblastoma multiforme A Masi et al allows the molecular identification of the ion channels responsible for the ion currents recorded by patch-clamp investigation. To address these issues, we performed an RT -PCR survey of the most likely candidate genes: Kir 2.1, a molecular component of I IRK , known to be highly expressed in cortical astrocytes (Olsen and Sontheimer, 2004), herg1, a molecular component of I hERG , the member of the herg family most frequently expressed by tumour cells and helk2. Representative examples of the PCR-amplified transcripts are reported in Figure 3.…”

“…The expression profile of ion channels is known to be profoundly altered in glial tumour cells (Bordey and Sontheimer, 1998;Bubien et al, 1999;Ranson and Sontheimer, 2001;Olsen and Sontheimer, 2004). In some contexts, these alterations have been shown to trigger relentless growth (reviewed in Arcangeli and Becchetti, 2006) and invasiveness (Soroceanu et al, 1999;Ishiuchi et al, 2002;Olsen et al, 2003).…”

“…The expression pattern of these two channels was compared to that of the classical inward rectifying (IRK) K þ channels (KCNJ2 or Kir 2.1), widely expressed in astrocytic cells, and whose plasma membrane expression is downregulated in gliomas (Olsen and Sontheimer, 2004).…”