Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis

Area of Science:

• * Cell biology
• * Molecular pharmacology
• * Physiology

Background:

• * Pulmonary endothelial cells regulate vascular tone and integrity via calcium signaling.
• * Store-operated calcium entry (I_soc) currents contribute to endothelial cell gap formation.
• * FKBP51 regulates I_soc, but its inhibitory mechanism remains unclear.

Purpose of the Study:

• * To investigate the role of Protein Phosphatase 5 (PP5C) in FKBP51-mediated I_soc inhibition.
• * To determine if PP5C is a component of the I_soc regulatory heterocomplex.
• * To elucidate PP5C's contribution to FKBP51's effect on endothelial cell barrier function.

Main Methods:

• * siRNA-mediated knockdown and genetic disruption of PP5C in pulmonary artery endothelial cells (PAECs) and HEK293 cells.
• * Reconstitution experiments with catalytically active or inactive PP5C.
• * Co-immunoprecipitation and cell fractionation to assess protein interactions and localization.
• * Measurement of inter-endothelial cell gap formation using microscopy and electric cell-substrate impedance sensing (ECIS®).

Main Results:

• * Suppression of PP5C attenuated FKBP51-mediated I_soc inhibition in both cell types.
• * Reintroduction of active PP5C restored FKBP51's inhibitory effect on I_soc.
• * PP5C co-precipitated with TRPC4, a subunit of the I_soc channel, and was localized to the same membrane fractions as the I_soc complex.
• * PP5C suppression negated the protective effect of FKBP51 against calcium entry-induced gap formation.

Conclusions:

• * PP5C is a novel component of the I_soc regulatory heterocomplex.
• * PP5C is required for FKBP51 to inhibit I_soc in pulmonary endothelial cells.
• * PP5C plays a critical role in maintaining endothelial barrier integrity by modulating I_soc.