Details

Autor(en) / Beteiligte

• Numata, Tomohiro
• Sato, Kaori
• Christmann, Jens
• Marx, Romy
• Mori, Yasuo
• Okada, Yasunobu
• Wehner, Frank

Titel

The [Delta]C splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation

Ist Teil von

• The Journal of physiology, 2012-03, Vol.590 (5), p.1121

Ort / Verlag

London: Wiley Subscription Services, Inc

Erscheinungsjahr

2012

Quelle

Wiley Online Library

Beschreibungen/Notizen

• 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 [Delta]C-splice variant as the molecular correlate of the HICC, which was supported by quantification of Ca2+ 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 [Delta]C-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and 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.