HMIT activity in HEK293T cells transiently expressing HMIT triple mutant at the plasma membrane. A HMIT triple mutant is expressed at the plasma membrane in HEK cells as indicated by co-localisation with pan-cadherin antibody (Abcam, 1:1000) observed by confocal microscopy, in contrast to wild-type HMIT, which is mainly localised intracellularly. B [3H]-myo-inositol uptake and its concentration dependent inhibition using the non-selective HMIT inhibitor phloridzin were demonstrated at pH 6.0. C Top left: schematic diagram illustrating the proposed electrogenic transport by HMIT. Top right: representative traces of inositol-evoked currents recorded in a HEK293T cell transiently transfected with HMIT triple mutant (HMIT-TM). Middle: concentration-response relationship for myo-inositol in the HMIT triple mutant at pH 5.2 (n = 5–9 cells per data point). Bottom: pH sensitivity of currents evoked by myo-inositol (2 mM), recordings were made in HEK293T cells transfected with the HMIT triple mutant (n = 4–7 per data point). D The HMIT triple mutant was also demonstrated to transport IP3. Similar to observations with myo-inositol, the electrogenic transport of IP3 was observed at pH 5.2 and not at pH 7.4 (n = 4–6 per data point).