Regulation of volume-activated chloride channels by P-glycoprotein: phosphorylation has the final say!

Abstract

P-glycoprotein (Pgp) is a transmembrane transporter causing efflux of a number of chemically unrelated drugs and is responsible for resistance to a variety of anticancer drugs during chemotherapy. Pgp overexpression in cells is also associated with volume-activated chloride channel activity; Pgp is thought to regulate such activity. Reversible phosphorylation is a possible mechanism for regulating the transport and chloride channel regulation functions of Pgp. Protein kinase C (PKC) is a good candidate for inducing such phosphorylation. Hierarchical multiple phosphorylation (e.g. of different serines and with different PKC isoforms) may shuttle the protein between its different states of activity (transport or channel regulation). Cell volume changes may trigger phosphorylation of Pgp at sites causing inhibition of transport. The possible regulation of chloride channels by Pgp and the potential involvement of reversible phosphorylation in such regulation is reviewed.

Figure 1. Schematic representation of Pgp

The Pgp molecule consists of two similar transmembrane domains TMD1 and TMD2, each followed by a cytoplasmic nucleotide binding domain (termed NBD1 and NBD2, respectively). The NBDs are responsible for the ATP hydrolysis that is associated with drug efflux. A short linker region joins NBD1 with TMD2. Four previously identified phosphorylation target sites for PKC and PKA within the linker region of Pgp are also shown.

Figure 2. Regulation of ATPase activity by PKC α-mediated phosphorylation

Pgp and PKC α were co-expressed in Sf9 cells as described in Ahmad et al. (1994). Phosphorylation of Pgp stimulates ATPase activity of Pgp in the presence or absence of verapamil. This stimulation is lost when serine 671 in the Pgp linker region is mutated to aspargine. Reproduced from Ahmad et al. (1994) with permission of the American Chemical Society.

Figure 3. Differential effects of PKA and PKC on the swelling-induced Cl⁻ current I_{Cl, swell} in MDR cells

A, activation of PKC with the phorbol ester PMA reduced rate of activation of I_{Cl, swell} but not its steady-state value. B, activation of PKA with 8-bromo-cyclic AMP (8-Br-cAMP) reduced steady-state current levels. C, simultaneous activation of both PKA and PKC with 8-Br-cAMP and PMA, generated a ‘combined’ effect, such that onset of current was delayed and final current magnitude was reduced. The experimental protocol was described in Vanoye et al. (1999). Reproduced from Vanoye et al. (1999) with permission of the American Physiological Society.