Characterization of a Digitonin‐Solubilized Bovine Brain H₃ Histamine Receptor Coupled to a Guanine Nucleotide‐Binding Protein

Abstract

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.

Correlation of apparent affinity values from H3‐receptor binding assays with apparent affinity (pKapp) and intrinsic activity (α) from functional bioassays

“…Therefore, according to this model, high‐affinity H 3 ‐receptor agonist binding could result from either binding to preformed high‐affinity receptor states (R* and R*G) or from the induction of these high‐affinity states through binding to low‐affinity receptors (R). Low‐affinity agonist binding in buffer B (0.07,0.1,0.1) can be explained by considering that, under these conditions, the agonist binds only to the low‐affinity receptor state (R) and cannot bind to or induce the formation of R* or R*G. Although, at the time these studies were performed it was not definitely known that the H 3 ‐receptor was G‐protein coupled because it had not yet been cloned, the possibility that agonists could induce H 3 ‐receptor ternary complex formation was supported by studies suggesting that these receptors were linked to effector systems through G‐proteins ( Arrang et al ., 1990 ; West et al ., 1990 ; Zweig et al ., 1992 ; Clark et al ., 1993 ; Litosch et al ., 1993 ; Clark and Hill, 1995 ; Clark and Hill, 1996 ).…”

Identification of a Histamine H3‐like Receptor in the Zebrafish (Danio rerio) Brain

“…[ 3 H]NAMH was chosen as the radioligand for H 3 receptor binding in the present study. This agonist has been widely used and proven to be suitable for H 3 receptor binding studies (Korte et al, 1990 ; Cumming et al, 1991 ; Zweig et al, 1991 ; Ryu et al, 1996). Another tritiated H 3 receptor agonist used in binding studies is ( R )α‐[ 3 H]methylhistamine (see, e.g., Pollard et al, 1993).…”

Evidence that histamine homologues discriminate between H3‐receptors in guinea‐pig cerebral cortex and ileum longitudinal muscle myenteric plexus

“…Recently, Lovenberg et al . (1999) reported the cloning and characterization of a human G‐protein‐coupled H 3 ‐receptor in agreement with some previous evidence to suggest that this receptor could be G‐protein‐coupled (Arrang et al ., 1990; Zweig et al ., 1992; Clark & Hill, 1995; 1996). Some compounds have been shown to directly activate G‐proteins (Detert et al ., 1995; 1996; Leschke et al ., 1997) and therefore we considered whether this action could explain the discrepancies observed for the homologues of histamine (VUF8326, VUF4701, VUF4702 and VUF4732).…”