Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles

Abstract

Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which when occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of [3H]vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclo-hexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. [3H]Vesamicol binding depends on deprotonation of a group of pKa1 = 6.26 +/- 0.03 and protonation of a group of pKa2 = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analogue dl-trans-4-oxo-4-[5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl )-1- naphthalenyl]amino]butanoic acid (TPNB) is an effective inhibitor of AcCh active transport with an IC50 value of (51 +/- 8) x 10(-9) M. At 23 degrees C, [3H]vesamicol bound to the receptor at a rate of (1.74 +/- 0.06) x 10(5) M-1 s-1, and excess unlabeled vesamicol displaced a low concentration of bound [3H]vesamicol at 0.29 +/- 0.01 min-1. At 0 degrees C, 10 microM unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound [3H]vesamicol at 0.16 +/- 0.02 min-1 and 64 +/- 2% at 0.013 +/- 0.001 min-1. One micromolar unlabeled vesamicol behaved similarly.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane Reorientation of the Substrate-Binding Site in Vesicular Acetylcholine Transporter

“…Postnuclear supernatant (50 µL) containing 200 µg of protein was mixed with 50 µL of UBB also containing 100 µM diethyl-p-nitrophenyl phosphate (UBB/P). Uptake was initiated by the addition of 100 µL of UBB/P containing 10 …”

Transport mechanisms in acetylcholine and monoamine storage

“…Because a change in either V max or K m alters transport rate in subsaturating substrate (as rate Ϸ [substrate o ]V max /K m ), these data do not determine whether V max or K m is affected by pH. Equilibrium binding of vesamicol or a high-affinity synthetic analog of ACh requires deprotonation with pK a 7.1 and 7.4, respectively (43)(44)(45). Vesamicol binding also requires protonation with pK a Ͼ 9.…”

Purification and Characterization of a Nonvesicular Vesamicol‐Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain

“…The homogenates were centrifuged at 4°C in a Beckman SW 25.1 rotor at 24,000 rpm (90,000 g,,,,,) for 22 h. The supernatants were assayed for protein content and vesamicol binding using 10 pM ['H]vesamicol by the centrifugation-gel filtration assay. The pellets were resuspended in 20 ml of the above buffer and assayed for protein content, vesamicol binding using 5 pM ['H]vesamicol and vesamicol binding after incubation with 100 nM (~)-4-aminobenzovesamicol' (vesamicol analogue 72, Table 2) for 60 min by a glass-fiber filter method (Kornreich and Parsons, 1988). Analogue 72 binds tightly enough to the vesicular receptor and dissociates slowly enough (halflife 5.4 h) that little vesicular receptor will become available to ['H]vesamicol during the course of the vesamicol binding assay, even though the [3H]vesamicol concentration is much greater than the analogue 72 concentration (Rogers and Parsons, 1990).…”

“…Vesamicol [(-)-truns-2-(4-phenylpiperidino)cyclohexanol] is a potent inhibitor of acetylcholine (ACh) active transport by synaptic vesicles (Anderson et al, 1983;Bahr and Parsons, 1986~;Rogers et al, 1989). The inhibition is mediated by a receptor located in the vesicle membrane (Bahr and Parsons, 1986b;Kornreich and Parsons, 1988). As a result of ACh storage inhibition, the drug inhibits evoked release of newly synthesized ACh from a wide variety of intact nerve terminal preparations (Marshall and Parsons, 1987 and references therein;Lupa, 1988;Mykita and Collier, 1989;Vizi, 1989;Marien and Richard, 1990;Unsworth and Johnson, 1990).…”