Signalling Events Employed in the Hypertonic Activation of Cation Channels in HeLa Cells

Wehner, Frank; Numata, Tomohiro; Subramanyan, Muthangi; Takahashi, Nobuyuki; Okada, Yasunobu

doi:10.1159/000104155

Cited by 8 publications

(9 citation statements)

References 51 publications

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“…1). Interestingly, membrane currents that are virtually identical to the above activation of hypertonicity‐induced cation channels (HICCs) – including a linear I–V relationship (Wehner et al 2003 b , 2007; Shimizu et al 2006; Numata et al 2007 b , 2008) – were emanating when, under isotonicity, 500 μ m cADPr was administered in the patch‐pipette (Fig. 1 and ), i.e.…”

Section: Resultsmentioning

confidence: 94%

“…To further test the working hypothesis that TRPM2 is indeed reflecting the HICC in HeLa cells, the pharmacology of cADPr‐induced currents was examined. 2‐Amino‐ethoxydiphenyl borate (2‐APB) and flufenamate (FFA) were employed, which are both established blockers of the HICC in this system (Wehner et al 2003 b , 2007; Shimizu et al 2006; Numata et al 2007 b , 2008). In Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

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The ΔC splice‐variant of TRPM2 is the hypertonicity‐induced cation channel in HeLa cells, and the ecto‐enzyme CD38 mediates its activation

Numata

Sato

Christmann

et al. 2012

The Journal of Physiology

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Key points• Hypertonicity-induced cation channels (HICCs) are key players in proliferation and apoptosis but their molecular identity has remained elusive.• We report that in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as activators of TRPM2 cation channels, elicited currents that are identical to the osmotic activation of HICCs.• siRNA-mediated silencing of the expression of TRPM2 and CD38 (as the supposed source of ADPr and cADPr) inhibited HICC and nucleotide-induced currents and the osmotic volume response of cells.• Quantification of intracellular cADPr and extracellular application of nucleotides revealed that the outwardly directed gradient rather than the intracellular activity of ADPr and cADPr is the triggering factor for TRPM2.• Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which was supported by quantification of Ca 2+ selectivity.• Immunoprecipation and FRET/FLIM assays revealed the interaction of TRPM2 and CD38 and we propose a transport-related nucleotide export via CD38 as a novel mechanism of TRPM2 activation.Abstract Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra-vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides. Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca 2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and K. Sato and J. Christmann contributed equally to this work. CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive. (

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

The ΔC splice‐variant of TRPM2 is the hypertonicity‐induced cation channel in HeLa cells, and the ecto‐enzyme CD38 mediates its activation

Numata

Sato

Christmann

et al. 2012

The Journal of Physiology

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“…S1). However, hypertonicity-induced cation channels can be excluded as a candidate because PI-3K inhibitors (wortmannin and LY294002) suppressed RVI without significantly affecting the hypertonicity-induced cation channel current in HeLa cells (21). Na…”

Section: Discussionmentioning

confidence: 99%

“…Volume after Osmotic Shrinkage-In our previous study, PI-3K inhibitors (wortmannin and LY294002) inhibited the induction of RVI in HeLa cells under hypertonic conditions (21). Therefore, in the present study, we first examined whether suppressing the activity of Akt, a downstream target of PI-3K, inhibits the induction of RVI.…”

Section: Hypertonic Challenge Activates Akt Inducing Recovery Of Cellmentioning

confidence: 94%

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Subramanyam¹,

Takahashi²,

Hasegawa

et al. 2010

Journal of Biological Chemistry

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Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/ Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-␣, or a Fas ligand inhibited both RVI and hypertonicityinduced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.

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“…The membrane has been treated as the key player in cell volume regulation [45–49]. There are many proposals for the existence of membrane based volume sensors such as stretch-activated ion channels, but there are no reliable mechanistic explanations for how cell volume, an extrinsic variable, is controlled by the intrinsic variable of membrane stress [48,50–52]. The data in this paper suggests that there may be no explicit molecular sensor for cell volume, any more than there is a molecular sensor for the volume of a sponge; the volume is set by a force balance.…”

Section: Discussionmentioning

confidence: 99%

Atomic force microscopy analysis of cell volume regulation

Spagnoli

Beyder

Besch³

et al. 2008

Phys. Rev. E

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Cells swell in response a hypoosmotic challenge. By converting osmotic pressure to hydrostatic pressure at the cell membrane via van't Hoff's law, and converting that to tension via Laplace's law one predicts that the cell membrane should stretch and become stiff. We tested this prediction using the atomic force microscopy. During osmotic swelling cells did not become stiff and generally became softer. This result contradicts the assumption of the cell membrane as the constraining element in osmotic stress but is consistent with the cytoskeleton acting as a cross-linked gel. Models of the cells' response to osmotic stress must include energy terms for three-dimensional stresses.

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Signalling Events Employed in the Hypertonic Activation of Cation Channels in HeLa Cells

Cited by 8 publications

References 51 publications

The ΔC splice‐variant of TRPM2 is the hypertonicity‐induced cation channel in HeLa cells, and the ecto‐enzyme CD38 mediates its activation

The ΔC splice‐variant of TRPM2 is the hypertonicity‐induced cation channel in HeLa cells, and the ecto‐enzyme CD38 mediates its activation

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Atomic force microscopy analysis of cell volume regulation

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