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

Abstract

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. (

Channels and Volume Changes in the Life and Death of the Cell

“…A study in HeLa cells (Wehner et al, 2006) showing RVI reduction by SKF-96365, a blocker of TRP channels, raised the question of whether the hypertonicity-induced cation channels are actually TRP channels, and recent evidence pointed to TRPM2 as the channel involved. A recent report, also in HeLa cells (Numata et al, 2012), showed that nucleotides adenosine diphosphate ribose (ADPr) and cyclic ADPr, reported as typical TRPM2 activators (Pedersen et al, 2005;Pedersen and Nilius, 2007), generate cation currents similar to those elicited by hypertonicity, previously known as hypertonicityactivated cation channels. In the same line, small-interfering RNA silencing of TRPM2 expression or the precursor enzyme of the TRPM2 nucleotide activators abolish the hypertonicityelicited cation current and mildly reduce RVI.…”

“…In the same line, small-interfering RNA silencing of TRPM2 expression or the precursor enzyme of the TRPM2 nucleotide activators abolish the hypertonicityelicited cation current and mildly reduce RVI. Cloning of TRPM2 identified the DC-splice variant as the molecular entity corresponding to the nucleotide-activated current (Numata et al, 2012), which suggests that TRPM2 may represent the molecular identity of the hypertonicityactivated cation channels. Another member of the TRP channel family, TRPV2, seems to participate in RVI via an interesting connection with the electroneutral cotransporter NKCC, which as previously mentioned is a main effector in RVI.…”

Transient Receptor Potential Channels as Drug Targets: From the Science of Basic Research to the Art of Medicine

“…A splice variant of TRPM2 has been reported as a cell-shrinking or hypertonicity-induced cation channel, which is functionally involved in apoptosis and cell proliferation (Numata et al, 2012).…”

Intracellular Calcium Concentrations Regulated by Cyclic ADP-Ribose and Heat in the Mouse Hypothalamus

“…However, the effect of cADPR is much weaker than that of ADPR (Numata et al, 2012). Very recently, it has been reported that the epithelium cell volume depends on both CD38 and TRPM2, after interaction with truncated TRPM2 to CD38 after dehydration (Numata et al, 2012). Therefore, it is of interest to examine whether such molecular interactions occur in the nerve cells of the hypothalamus.…”

“…cADPR was also known to the same bind site of TRPM2 channels and further modulates them. However, the effect of cADPR is much weaker than that of ADPR (Numata et al, 2012). Very recently, it has been reported that the epithelium cell volume depends on both CD38 and TRPM2, after interaction with truncated TRPM2 to CD38 after dehydration (Numata et al, 2012).…”