FKBP12-BOUND CALCINEURIN CONTROL TMEM16A ACTIVITY

Abstract

The Ca2+-dependent Cl- channel TMEM16A activity is vital for mammals. Critical functions such as blood pressure, pain, fluid and electrolyte secretion, peristalsis, and electrical activity depend on TMEM16A Cl- fluxes. Ciclosporin A (CsA) and FK506, two immunosuppressants that inhibit calcineurin (CaN), down-regulate TMEM16A function. However, the underlying mechanism remains undetermined. CsA binds to cyclophilin 1, whereas FK506 binds to FKBP12, and both complexes subsequently inhibit CaN, a phosphatase that maintains TMEM16A function. Here, we show that TMEM16A-EGFP and mRFP-FKBP12 colocalize in HEK-AD293 cells stimulated with the Ca2+ ionophore ionomycin, FKBP12 co-immunoprecipitated with TMEM16A expressed in CaN-depleted HEK-AD293 cells, ionomycin favoured TMEM16A and FKBP12 interaction in a bimolecular fluorescence complementation assay in live cells, and TMEM16A currents recorded from CaN-depleted HEK-AD293 cells were insensitive to CsA and FK506. These data support the idea that FKBP12 works as an auxiliary protein of TMEM16A, thus enabling the interaction of CaN under physiological conditions. This heteromerization is essential to sustain TMEM16A activity. On the contrary, disrupting the Ca2+-stimulated CaN-FKBP12-TMEM16A heterotrimer or inhibiting CaN with CsA or FK506 does not preclude TMEM16A activation but decreases activity.