The voltage sensitive phosphoinositide (PI) phosphatases (VSPs) and the PI-phosphatase PTEN are members of the PTP phosphatase superfamily, sharing their CX5R signature motif. PTEN and VSPs differ in substrate specificity, PTEN being a PI(3,4)P2/PI(3,4,5)P3-3-phosphatase, whereas VSPs are PI(4,5)P2/PI(3,4,5)P3-5-phosphatases. The most striking difference between both classes of phosphatases lies in the CX5R motif itself, were an alanine in PTEN is replaced by a glycine in VSP. But this amino acid exchange is insufficient to fully explain the difference in specificity (Leitner et al., Biophysical Journal 102(3) p. 246a). Another notable difference between PTEN and VSPs is the absence of the TI-loop motif of PTEN in VSPs, where it is replaced by the ET-motif of the “gating-loop” (Liu, Kohout et al., NSMB 2012).We set out to elucidate the significance of the TI/ET motif, using a set of voltage sensitive phosphatases: Ci-VSP and two engineered VSPs, PTENCiV and hVSP1CiV. The latter contain Ci-VSP's voltage sensing domain and the phosphatase domain of PTEN or hVSP1 (= TPTE2). The effects of TI/ET (in PTEN) or ET/TI (in VSPs) mutations were studied using fluorescent PI sensor domains for the detection of phosphatase activity.We find that the TI/ET mutation transforms PTEN into a PI(3,4,5)P3-5-phosphatase with no detectable PI(4,5)P2-phosphatase activity. An additional A/G -mutation in the CX5R-motif is required to induce PI(4,5)P2-5-phosphatase activity, thereby converting PTEN's phosphatase phenotype into that of VSPs. For VSPs the situation appears to be more complex, as we failed to convert their phenotype from 5- to 3-phosphatase by mirroring these mutations (i.e. ET/TI and G/A).This work was supported by the University Medical Center Giessen and Marburg (grant UKGM 32/2011 MR) to C.R.H. and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.