The multidrug resistance P‐glycoprotein (P‐gp), which transports hydrophobic drugs out of cells, is also associated with volume‐activated chloride currents. It is not yet clear whether P‐gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P‐gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume‐activated chloride currents indistinguishable from those obtained in P‐gp‐expressing cells except that they were insensitive to PKC. HeLa cells did not express detectable P‐gp but, following transient transfection with cDNA encoding P‐gp, the volume‐activated channels acquired PKC regulation. PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P‐gp were replaced with alanine. Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation. These data demonstrate that PKC‐mediated phosphorylation of P‐gp regulates the activity of an endogenous chloride channel and thus indicate that P‐gp is a channel regulator.

The spo-87 mutation is one of two sporulation mutations originally used to define the spUJ locus of Bacillus subtiIis. We now show that it blocks sporulation after completion of prespore engulfment (stage 111). Surprisingly, the operon is expressed vegetatively, probably from a @*-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation. DNA sequencing reveals that the locus defined by sp0-87, which we now designate spIIIJ, consists of a bicistronic operon. However, only the first gene is essential for sporulation; the function of the second cistron is cryptic. The predicted SpoIIIJ product has an M, of 29409. It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids. Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the nC form of RNA polymerase but not transcription of the spoIIIG gene encoding oc. The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.

P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [ 3 H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.