MUSCARINIC ACETYLCHOLINE-RECEPTOR TRAFFICKING IN STREPTOLYSIN O-PERMEABILIZED MDCK CELLS

We investigated the validity of streptolysin O (SLO)-permeabilized Mad in-Darbin canine kidney (MDCK) cells which express muscarinic acetylch oline receptors (mAChRs) coupled to pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) for the study of the molecula r machinery that regulates mAChR internalization and recycling. Exposu re of SLO-permeabilized cells to carbachol-reduced cell surface recept or number by up to 40% without changing total receptor number The kine tics and maximal extent of receptor internalization as well as the pot ency of carbachol to induce receptor internalization were almost ident ical in SLO-permeabilized and non-permeabilized cells. Using this semi -intact cell system, we studied the effect of various agents affecting components potentially involved in receptor trafficking. Internalizat ion was prevented by treatment of the SLO-permeabilized MDCK cells wit h (i) the stable ATP analogues, adenosine 5'-O-(3-thiotriphosphate) an d adenylylimidodiphosphate, to block ATP-dependent processes, and (ii) heparin to block G protein-coupled receptor kinases. Inclusion of the stable GTP analogue, guanosine 5'-O-(3-thiotriphosphate), increased t he rate but not the extent of receptor internalization. None of the me mbrane-impermeant agents affected receptor internalization in intact M DCK cells. This model system also allowed recycling of internalized re ceptors back to the plasma membrane. After removal of the agonist, cel l surface receptor number in SLO-permeabilized cells returned to contr ol values within 90 min with the same kinetics as seen in intact cells . Inclusion of guanosine 5'-O-(3-thiotriphosphate) shortened the recov ery time. These data suggest that both ATP-dependent kinases including G protein-coupled receptor kinases and G proteins participate in rece ptor internalization and recycling. In summary, the (-)SLO-permeabiliz ed MDCK cell is a feasible model system for the study of mAChR interna lization and recycling and allows manipulation of the intracellular mi lieu with membrane-impermeable macromolecules.