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Identification of residues contributing to the subtype selectivities of gallamine and UH-AH 37 at muscarinic receptors
Vol. 60, Nos. U/14,1997
1173
Abstracts
21 IDENTlFlCATlON OF GALLAMINE A.L. Gnagey ‘Departments
OF RESIDUES CONTRlBUTlNG TO THE SUBTYPE SELECTIVITIES AND UH-AH 37 AT MUSCARINIC RECEPTORS.
and J. Ellis. Department of Psychiatry, University of Vermont, Burlington VT and of Psychiatry and Pharmacology, Penn State University, Hershey PA 17033 USA
Gallamine is the prototypical muscarinic allosteric ligand and has a marked preference for the m2 subtype. UH-AH 37 is a competitive ligand related to pirenzepine that has lower affinity at the m2 receptor, compared to the other four subtypes. Nonetheless, the m2/m5 subtype selectivities of both of these ligands have previously been attributed to a short (31 amino acid) segment of the receptor sequence, predicted to make up most of the sixth transmembrane domain and all of the third outer loop of the receptor structure”. We have used site-directed mutagenesis to change the amino acid residues in this stretch of the m5 receptor to the corresponding amino acids of the m2 receptor one at a time; in the two cases where there were adjacent residues to be mutated, we also prepared the corresponding double mutation. None of these mutations produced marked changes in the affinity or rate of dissociation of the labeled ligand, [3H]NMS. For UH-AH 37, only one mutation (thr to ala at m545s)resulted in a reduction of affinity similar to that seen when the entire segment was substituted. Surprisingly, two mutations led to enhancements of affinity for gallamine that were similar to that seen when the entire segment was substituted. These were lys to pro at m5470and val to asn at m5474. However, the double mutant asp-lys to ala-pro at m5469d7a did not enhance the affinity for gallamine, leading us to believe that the enhancement of affinity produced by the single substitution at m5470was due to an anomalous interaction with asp&‘, which is present in the wild-type m5, but not at the corresponding position in m2. Thus, the lower affinity of UH-AH 37 for the m2 receptor appears to be due to the lack of the threonine that is present at m545s (and also at the corresponding location in the ml, m3, and m4 subtypes). On the other hand, the higher affinity of gallamine for the m2 receptor appears to be due to an interaction with the asparagine at m24’g, which does not occur at the corresponding position in m5 or in any of the other subtypes. ‘Wess et al., Mol. Pharmacol. 41369 ‘Ellis et al., Mol. Phatmacol. 44583 [Supported by 1701 AGO52141
22 A COMMON SITE OF ACTION FOR TWO ALLOSTERK
ANTAGONISTS.
A. Christopoulos, A. Lanzafame and F. Mitchelson, Dept. of Pharmaceutical Biology Pharmacology, Victorian College of Pharmacy (Monash University), Melbourne, Australia.
and
Combination of two competitive antagonists produces additive dose ratios when tested with an agonist in functional experiments, whereas the allosteric antagonist heptane-1,7-bis(dimethyl-3’phthalimidopropyl) ammonium bromide (C$3’-phth), in combination with a number of competitive antagonists, produces supra-additive dose ratios (Christopoulos & Mitchelson, 1994). To test whether Cr/3’-phth acts at a common site to the allosteric antagonist gallamine, experiments with combinations of the antagonists were conducted on the negative inotropic response to carbachol in guinea pig atria. Combination of C7/3’-phth (10 @i) with the competitive antagonist, a&opine (0.2 PM), produced a dose-ratio which was ea. 6-fold greater than expected if both antagonists were acting competitively. The combination of gallamine (100 PM) with atropine gave a combined doseratio which was close to that expected for two competitive antagonists, although with acetylcholine as agonist the combined dose ratio was cu. 66% of that produced by atropine alone. Combination of CT/~‘-phth (10 PM) with gailamine (100 PM), using carbachol as the agonist, gave a combined dose ratio which was less than that expected for two competitive antagonists. The findings could be explained with a model involving C7/3’-phth and gallamine competing for a single site with cooperativity factors (01) of cu. 1000 and 200 respectively for their interaction with carbachol at the alfosteric site; values of a similar order for the co-operativity factors were obtained from separate experiments investigating the effect of each antagonist alone on responses to carbachol. Christopoulos
A. and F. Mitchelson (1994) Mol. Pharmacol., 46, 105-l 14.
1173
Abstracts
21 IDENTlFlCATlON OF GALLAMINE A.L. Gnagey ‘Departments
OF RESIDUES CONTRlBUTlNG TO THE SUBTYPE SELECTIVITIES AND UH-AH 37 AT MUSCARINIC RECEPTORS.
and J. Ellis. Department of Psychiatry, University of Vermont, Burlington VT and of Psychiatry and Pharmacology, Penn State University, Hershey PA 17033 USA
Gallamine is the prototypical muscarinic allosteric ligand and has a marked preference for the m2 subtype. UH-AH 37 is a competitive ligand related to pirenzepine that has lower affinity at the m2 receptor, compared to the other four subtypes. Nonetheless, the m2/m5 subtype selectivities of both of these ligands have previously been attributed to a short (31 amino acid) segment of the receptor sequence, predicted to make up most of the sixth transmembrane domain and all of the third outer loop of the receptor structure”. We have used site-directed mutagenesis to change the amino acid residues in this stretch of the m5 receptor to the corresponding amino acids of the m2 receptor one at a time; in the two cases where there were adjacent residues to be mutated, we also prepared the corresponding double mutation. None of these mutations produced marked changes in the affinity or rate of dissociation of the labeled ligand, [3H]NMS. For UH-AH 37, only one mutation (thr to ala at m545s)resulted in a reduction of affinity similar to that seen when the entire segment was substituted. Surprisingly, two mutations led to enhancements of affinity for gallamine that were similar to that seen when the entire segment was substituted. These were lys to pro at m5470and val to asn at m5474. However, the double mutant asp-lys to ala-pro at m5469d7a did not enhance the affinity for gallamine, leading us to believe that the enhancement of affinity produced by the single substitution at m5470was due to an anomalous interaction with asp&‘, which is present in the wild-type m5, but not at the corresponding position in m2. Thus, the lower affinity of UH-AH 37 for the m2 receptor appears to be due to the lack of the threonine that is present at m545s (and also at the corresponding location in the ml, m3, and m4 subtypes). On the other hand, the higher affinity of gallamine for the m2 receptor appears to be due to an interaction with the asparagine at m24’g, which does not occur at the corresponding position in m5 or in any of the other subtypes. ‘Wess et al., Mol. Pharmacol. 41369 ‘Ellis et al., Mol. Phatmacol. 44583 [Supported by 1701 AGO52141
22 A COMMON SITE OF ACTION FOR TWO ALLOSTERK
ANTAGONISTS.
A. Christopoulos, A. Lanzafame and F. Mitchelson, Dept. of Pharmaceutical Biology Pharmacology, Victorian College of Pharmacy (Monash University), Melbourne, Australia.
and
Combination of two competitive antagonists produces additive dose ratios when tested with an agonist in functional experiments, whereas the allosteric antagonist heptane-1,7-bis(dimethyl-3’phthalimidopropyl) ammonium bromide (C$3’-phth), in combination with a number of competitive antagonists, produces supra-additive dose ratios (Christopoulos & Mitchelson, 1994). To test whether Cr/3’-phth acts at a common site to the allosteric antagonist gallamine, experiments with combinations of the antagonists were conducted on the negative inotropic response to carbachol in guinea pig atria. Combination of C7/3’-phth (10 @i) with the competitive antagonist, a&opine (0.2 PM), produced a dose-ratio which was ea. 6-fold greater than expected if both antagonists were acting competitively. The combination of gallamine (100 PM) with atropine gave a combined doseratio which was close to that expected for two competitive antagonists, although with acetylcholine as agonist the combined dose ratio was cu. 66% of that produced by atropine alone. Combination of CT/~‘-phth (10 PM) with gailamine (100 PM), using carbachol as the agonist, gave a combined dose ratio which was less than that expected for two competitive antagonists. The findings could be explained with a model involving C7/3’-phth and gallamine competing for a single site with cooperativity factors (01) of cu. 1000 and 200 respectively for their interaction with carbachol at the alfosteric site; values of a similar order for the co-operativity factors were obtained from separate experiments investigating the effect of each antagonist alone on responses to carbachol. Christopoulos
A. and F. Mitchelson (1994) Mol. Pharmacol., 46, 105-l 14.