Solubilized musarinic recognition sites from rat brain and heart: evidence in favor of a homogeneous population of sites

The binding characteristics of muscarinic receptors (MAChR) from rat brain and heart (membrane (m-) and 0.5% digitonin solubilized (s-)) were studied at 40C. [3H](-)QNB possessed the same affinity to both m- and s-MAChR (Kd = 20 pM). Pirenzepine (PZ) discriminated two affinities for brain m-MAChR and revealed a single low affinity in heart m-MAChR. S-MAChR from brain and heart displayed similar affinities for PZ ( Ki = 10 and 15 nM). High affinity [3H]PZ binding was also found for s-MAChR from both tissues. The receptor affinity for carbachol (CARB) was decreased after solubilization. There was a decrease in the proportion of high affinity state (from 40% to 20% in the brain and from 60% to 15% in the heart) with a 4-fold decrease in the high affinity Ki for CARB. Gpp(NH)p no longer had an effect on the CARB/[3H](-)QNB competition in s-MAChR. Increasing the temperature to 370C caused a 3-6 fold decrease in PZ's affinity to both m- and s-MAChR without altering the ratio of high/low affinity sites. The reduction of PZ's affinity was completely reversible with temperature in m-MAChR and partially reversible in s-MAChR. The authors results suggests that a homogeneous muscarinic recognition site has been solubilized. Solubilization with digitonin results in a separation of the binding site from effector systems and membrane constituents responsible for the tissue specific receptor heterogeneity