Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells

Abstract

The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca(2+), Mn(2+) and Na(+), have a high selectivity for Na(+) compared with Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 1 microM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca(2+) concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)(+) RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca(2+) inflow and a modest enhancement of thapsigargin-initiated Ca(2+) inflow (measured using fura-2) and no enhancement of the highly Ca(2+)-selective store-operated Ca(2+) current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na(+) and Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 3 microM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na(+) compared with Ca(2+); (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca(2+)-selective store-operated Ca(2+) channels.

The Mechanosensitive Ca2+ Channel as a Central Regular of Prostate Tumor Cell Migration and Invasiveness

“…Based on this apparent lack of TRPC1-linked SOC activity, Brereton et al (2000) proposed that TRPC1 might form the endogenous cation channel activated by the marine toxin, maitotoxin (MTX). However, in another study directly comparing the properties of the endogenous MTX-activated conductance measured in normal liver cells and 7 TRPC Family of Ion Channels and Mechanotransduction 135 the enhanced MTX-activated conductance measured in hTRPC1-transfected liver cells, Brereton et al (2001) found that the endogenous conductance showed a higher selectivity for Na + over Ca 2+ , and a higher sensitivity to Gd 3+ block (K 50% block = 1 µM vs 3 µM) compared with the enhanced conductance. These differences may indicate that other endogenous TRPC subunits combine with TRPC1 to form the endogenous MTX-activated conductance, whereas the enhanced MTX-activated conductance is formed exclusively by hTRPC1 homotetramers (Brereton et al 2001).…”

“…However, in another study directly comparing the properties of the endogenous MTX-activated conductance measured in normal liver cells and 7 TRPC Family of Ion Channels and Mechanotransduction 135 the enhanced MTX-activated conductance measured in hTRPC1-transfected liver cells, Brereton et al (2001) found that the endogenous conductance showed a higher selectivity for Na + over Ca 2+ , and a higher sensitivity to Gd 3+ block (K 50% block = 1 µM vs 3 µM) compared with the enhanced conductance. These differences may indicate that other endogenous TRPC subunits combine with TRPC1 to form the endogenous MTX-activated conductance, whereas the enhanced MTX-activated conductance is formed exclusively by hTRPC1 homotetramers (Brereton et al 2001). Finally, unlike in hTRPC1-transfected oocytes, hTRPC1-transfected rat liver cells did show an increased thapsigarininduced Ca 2+ inflow (Brereton et al 2000(Brereton et al , 2001).…”

“…In particular, low TRPC3 expression results in increased SOC activity, while high TRPC3 expression results in increased ROC activity (Vazquez et al 2003). This variation may occur because high expression levels favor TRPC3 homotetrameric channels, whereas low TRPC3 expression allows for heterotetrameric channels with incorporation of endogenous subunits as well as exogenous TRPC (Brereton et al 2001;Vazquez et al 2003). Differences in channel function may also arise depending upon whether the cell is permanently or transiently transfected, presumably because stable transfection provides added time for adaptive changes in endogenous protein expression (Lièvremont et al 2004).…”

Developmental Biology of Neoplastic Growth

“…Several cations like La 3+ , Gd 3+ , Ni 2+ and Ca 2+ have been shown to block MTX-elicited currents. Furthermore, several reports have suggested that MTX targets members of the transient receptor potential (TRP) family of channels in several cell types, notably TRPC1 (Brereton et al 2001;Chen and Barritt 2003;Trevino et al 2006). In addition, NSCC inhibitor of TRP channels, including di-and tri-valent cations that block MTX currents also block TRP channels (Clapham 2007).…”

Measuring Ca2+influxes of TRPC1-dependent Ca2+channels in HL-7702 cells with Non-invasive Micro-test Technique

“…No voltage-operated Ca 2+ channels (VOCCs) have been detected [5][6][7][8] . There is increasing evidence that members of the canonical subgroup of Transient Receptor Potential (TRP) proteins constitute tetramers of both receptoractivated and store-operated Ca 2+ channels (SOCs) [9][10][11] , and Transient Receptor Potential Canonical 1 (TRPC1) is considered as one of the most likely candidates in forming Ca 2+ channels in mammalian cells [12][13][14][15] . The Non-invasive Micro-test Technique (NMT) was developed in the late 20th century, and is a new technology for obtaining dynamic information on specific ionic/molecular activities on material surfaces, non-invasively.…”