Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP(2) in dorsal root ganglia neurons

BACKGROUND: Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP(2))-specific phosphatase (Inp54p) system to deplete PIP(2) in nociceptive dorsal root ganglia (DRG) neurons. RESULTS: We genetically targeted membrane-tethered CFP-FRB(PLF) (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRB(PLF) knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP(+) DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP(2), as measured with a PIP(2) biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRB(PLF)-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP(2)-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRB(PLF) in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRB(PLF) with endogenous FKBP12. CONCLUSIONS: Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP(2) in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRB(PLF), hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.