The ΔC splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation.
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 ΔC-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.
Department of Cell Physiology, National Institute of Physiological Sciences, Okazaki 444–8585, Japan.
Tomohiro
T
Numata
Kaori
K
Sato
Jens
J
Christmann
Romy
R
Marx
Yasuo
Y
Mori
Yasunobu
Y
Okada
Frank
F
Wehner
D051997:ADP-ribosyl Cyclase 1 / Q000502:physiology*; D000246:Adenosine Diphosphate Ribose / Q000502:physiology*; D049109:Cell Proliferation; D020868:Gene Silencing; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology*; D009243:NAD / Q000502:physiology; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D050053:TRPM Cation Channels / Q000502:physiology*
D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1