Receptor subtype | Experiment | Sequestration | |||
---|---|---|---|---|---|
pRK5 | RhoA | ||||
Dimethyl sulfoxide | Cytochalasin B | Dimethyl sulfoxide | Cytochalasin B | ||
m1 | 1 | 44% (113)1-a | 45% (125) | 26% (32) | 22% (27) |
2 | 46% (103) | 44% (138) | 24% (26) | 26% (32) | |
m2 | 3 | 69% (68) | 73% (80) | 26% (19) | 31% (18) |
4 | 73% (82) | 74% (86) | 20% (24) | 30% (23) | |
5 | 74% (93) | 74% (89) | 36% (20) | 41% (22) |
HEK-293 cells transiently transfected with m1 or m2 mAChR pCD-PS, along with pRK5 RhoA or control pRK5 were pretreated with 5 μg/ml cytochalasin B or vehicle (0.2% dimethyl sulfoxide) for 10 min at 37°C. Thereafter, cells were incubated with or without 1 mM carbachol for 60 min at 37°C in the continuing presence of cytochalasin B or vehicle. After washing, cell surface mAChR was determined by specific [3H]NMS binding to intact cells. Each experiment represents an independent set of transfections with pRK5 RhoA and control pRK5 DNA with the binding experiments done in duplicate.
↵1-a Values in parentheses, cell surface receptor number in untreated cells expressed in fmol/mg protein.