Diffusion delays and unstirred layer effects at monolayer cultures of Chinese hamster ovary cells: radioligand binding, confocal microscopy, and mathematical simulations.

Cells grown in monolayer culture offer a convenient system for binding and other experiments under conditions that preserve the complexity of the living state. Kinetics experiments, however, may be distorted by the time course of drug penetration into even so simple a "tissue" as the monolayer. The impediments include unstirred layers both above and between the cells, the congregation of receptors within the confined space between cells, and nonspecific binding to membrane components.

Kinetics of beta-funaltrexamine binding to wild-type and mutant mu-opioid receptors expressed in Chinese hamster ovary cells.

The two-stage reaction whereby the antagonist beta-funaltrexamine (beta-FNA) binds covalently to micro opioid receptors makes it a highly discriminating probe into the tertiary structure of the receptor's recognition pocket. To obtain a quantitative measure of how well this pocket is preserved in a mutated form of the receptor, in which His-297 is substituted with glutamine, we employed [3H]-beta-FNA to evaluate the kinetic rate constants for both the reversible as well as the irreversible stages of its binding to wild-type and mutant H297Q micro receptors stably expressed in Chinese hamster ovary cells. The expression levels of the wild-type and mutant H297Q receptors were matched by exploiting the variation in receptor density as a function of plating day and by raising the expression level by pretreatment with naloxone.

beta-Funaltrexamine, a gauge for the recognition site of wildtype and mutant H297Q mu-opioid receptors.

The antagonist beta-funaltrexamine (beta-FNA), known to bind covalently to mu-opioid receptors by a two-step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q mu-opioid receptors. We addressed whether this mutation, which enhances the intrinsic activities of alkaloid mu-receptor agents, affects both the reversible and covalent phases of beta-FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site.