Temperature effect on the detection of muscarinic receptor-G protein interactions in ligand binding assays

The ability of guanine nucleotides to lower agonist binding affinity provides a convenient indication of receptor-G protein coupling: guanine nucleotides convert muscarinic receptors from high-affinity states for agonists to low-affinity states. We studied the influence of assay temperature on the demonstration of this coupling in rat brainstem and atrium. Agonist affinity of brainstem receptors increased as temperature was lowered, reflecting a greater proportion of receptors in high-affinity conformations. The influence of 5'-guanylylimidodiphosphate, a stable analog of GTP, on agonist binding, determined directly (using [3H]oxotremorine-M) or indirectly (in [3H]N-methylscopolamine/carbamylcholine competition studies), was greatest from 16 to 20 degrees. Guanine nucleotide sensitivity was much reduced at 0-4 degrees and 37 degrees. Brainstem and atrial muscarinic receptors were similarly affected by temperature. We suggest that high-affinity receptor-G protein complexes are unstable at high temperatures, thereby decreasing agonist affinity and masking the guanine nucleotide effect. At low temperatures, the receptor-G protein complex is stabilized and fails to dissociate in the presence of guanine nucleotides. The optimum temperature for monitoring receptor-G protein interactions in binding assays was 16-20 degrees.