TRPM2 is a Ca2+-permeable cation channel that belongs to the M subfamily of Transient Receptor Potential (TRP) channel proteins. TRPM2 is a homotetramer, each monomer contains a cytosolic N-terminal TRPM-homology region, six transmembrane helices with an architecture typical to that of voltage-gated cation channels, and a cytosolic coiled-coil region followed by a NUDT9-Homology (NUDT9H) domain at the C-terminus.TRPM2 activates in the presence of Ca2+, ADP-Ribose (ADPR), and PIP2 (Toth and Csanady, 2012). ADPR binds to the NUDT9H-domain which displays ∼35% identity with NUDT9, a monomeric mitochondrial ADPR-pyrophosphatase with available crystal structure (Shen et al., 2003). At present, it is unclear whether NUDT9H is an active enzyme itself, or just an ADPR-binding domain. In contrast to the extremely high solubility of NUDT9, NUDT9H solubility is very low: when overexpressed in bacterial systems it forms inclusion bodies, and reprecipitates upon removal of chaotropic agents. This might be a consequence of the tetrameric nature of TRPM2, in the context of which it is unclear how the four N-terminal domains and the four NUDT9H domains interact with each other and/or with the transmembrane segments. The fact that ADPR binding to NUDT9H is necessary for channel activation suggests an extended interface between NUDT9H and some partnering segment within the complex. Surface exposure of this interface region may account for the reduced solubility of NUDT9H when expressed in isolation.Here we explore this hypothesis by expressing soluble NUDT9, insoluble NUDT9H, as well as different NUDT9/NUDT9H chimeras. By monitoring the solubility of the chimeras, we attempt to narrow down the search for the functional interface of NUDT9H. In addition, we try to identify the minimal substitutions required for increasing NUDT9H solubility. Finally, we use soluble NUDT9 to test hydrolysis of various substrates/ligands that activate the TRPM2 channel.