Muscarinic acetylcholine receptor trafficking in streptolysin O-permeabilized MDCK cells

We investigated the validity of streptolysin O (SLO)-permeabilized Madin-Darbin canine kidney (MDCK) cells which express muscarinic acetylcholine receptors (mAChRs) coupled to pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) for the study of the molecular machinery that regulated mAChR internalization and recycling. Exposure of SLO-permeabilized cells to carbachol-reduced cell surface receptor number by up to 40% without changing total receptor number. The kinetics and maximal extent of receptor internalization as well as the potency of carbachol to induce receptor internalization were almost identical 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. Internalization was prevented by treatment of the SLO-permeabilized MDCK cells with (i) the stable ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and 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 the rate but not the extent of receptor internalization. None of the membrane-impermeant agents affected receptor internalization in intact MDCK cells. This model system also allowed recycling of internalized receptors back to the plasma membrane. After removal of the agonist, cell surface receptor number in SLO-permeabilized cells returned to control values within 90 min with the same kinetics as seen in intact cells. Inclusion of guanosine 5'O-(3-thiotriphosphate) shortened the recovery time. These data suggest that both ATP-dependent kinases including G protein-coupled receptor kinases and G proteins participate in receptor internalization and recycling. In summary, the SLO-permeabilized MDCK cell is a feasible model system for the study of mAChR internalization and recycling and allows manipulation of the intracellular milieu with membrane-impermeable macromolecules.