Transient Receptor Potential Vanilloid subfamily member 6, TRPV6, is a highly Calcium selective channel, predominantly expressed in small intestine, placenta and exocrine tissues, but also correlated with tumor progression. Apart from a preferential conduction of Ca2+, TRPV6 and the related TRPV5 deviate from other TRP ion channels in terms of their constitutive activity. Despite the latter, TRPV6 function underlies modulation by several factors to ensure regulation of Ca2+ uptake and to preserve physiological levels. On the one hand, permeation is restricted by means of fast and slow Ca2+ dependent inactivation, FCDI and SCDI, respectively. On the other hand, binding of lipids, such as PIP2 and cholesterol, seems to maintain the conductive state of the channel. Taking advantage of electrophysiological recordings and site-directed mutagenesis, we thus investigated the effect of a series of TRPV6 residues that seem, based on structural data, to be involved in scaffolding a PIP2 binding site. We examined mutants that have been reported to either reveal a higher affinity for PIP2 or to be compromised in PIP2 binding. Furthermore, we analyzed a possible effect on SCDI by cholesterol. Reducing the availability of cholesterol in the plasma membrane by adding cholesterol oxidase, filipin or methyl-?-cyclodextrin showed no significant effect on SCDI. On the contrary, several TRPV6 mutants with alterations in potential PIP2 binding sites showed distinct levels of SCDI.