Acid-sensing Ion Channels in Malignant Gliomas

Abstract

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either ␥-or ␦-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxinbinding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na ؉ permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.

Glioma-specific Cation Conductance Regulates Migration and Cell Cycle Progression

“…Cells can also control their internal volume by regulating transport of Na ϩ , K ϩ , and Cl Ϫ across the plasma membrane, swelling or shrinking as required. Although much less sensitive to amiloride than the prototypical renal sodium channel ␣␤␥ENaC (K i amil Ͻ0.1 M (32)), the glioma cation channel that we have identified is critically involved in migration and proliferation (13)(14)(15)(16). This channel is inhibited by amiloride and its analogs and is blocked by PcTX-1, to which it is exquisitely sensitive (K i amil 10 -100 M; K i PcTX-1 K i ϳ40 pM (17)).…”

“…Cell lysates were homogenized by passing 10 times through a 22-gauge needle and centrifuged (13,200 rpm for 30 min at 4°C). Protein concentration of the supernatant was measured using BCA protein assay (Thermo-Scientific), and 25-50 g of protein lysates were used per lane for SDS-PAGE and immunoblotting.…”

“…We have previously shown that malignant glioma cells express an inward Na ϩ current that is not detected in normal astrocytes or low grade gliomas and is blocked by the diuretic amiloride (13). This cation current is due to expression of a cross-clade channel composed of members of the Deg/ENaC channel superfamily, i.e.…”

“…This cation current is due to expression of a cross-clade channel composed of members of the Deg/ENaC channel superfamily, i.e. acid-sensitive ion channel 1 (ASIC1) and two members of the epithelial sodium channel family, ␣-and ␥ENaC (13)(14)(15)(16). The amiloride-sensitive cation conductance found in glioma cells is also blocked by psalmotoxin-1 (PcTX-1), a peptide isolated from West Indies tarantula Psalmopoeus cambridgei (17,18), and is a highly specific blocker of ASIC1.…”

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

“…Thus, pH changes can be coupled to two important second messengers. It is also known that certain ion channels are pH sensitive (Akopian et al, 2000;Berdiev et al, 2003), and H + -V-ATPase could exert its effect by controlling another electrogenic component of LR patterning. There is also the possibility that activity of the asymmetrically localized H + -VATPase could cause redistribution of determinants (which may include the H + ion itself) by virtue of electrophoretic forces driven by the strong voltage gradients that the H + -V-ATPase can produce.…”