Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine
Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.
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- [Show abstract] [Hide abstract] ABSTRACT: By osmotic lysis in the presence of urea ghosts (60-100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4-6 mumol catecholamine/mg prot.) of the bovine medulla. In the presence of 1-300 mumol/l 3H-catecholamine and ATP-Mg2+, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (delta p). The same uptake was found by 3H-counting or by fluorimetric measurements. At various pH-values (pH 6.2-8.2) the Km and Vmax of the ATP-stimulated rate of uptake of 3H-catecholamine into ghosts was determined (at 30 degrees C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (delta pH = pHout - pHin) and the electrical potential difference (delta psi) were determined to calculate delta p under conditions of 3H-catecholamine uptake. When the pHout was increased (pH 6.2, 7.4, 8.2), the apparent Km of uptake decreased (50, 5, 1-2 mumol/l), showing a linear relation between pH and logarithm of Km. The Km was calculated for the uncharged catecholamine (with pK1 = 8.8 and pK2 = 10.0); it was nearly pH-independent and amounted to about 0.2 mumol/l. The Vmax declined only in the extreme pH-range. Between pH 6.6 and 7.8 Vmax and delta p showed a slight increase from 16 to 20 nmoles/(mg prot. X min) and from 110 to 140 mV, resp. In the same pH-range the pHin inside ghosts increased from pH 5.2 to 5.7, whereas delta psi was constant (30 mV). At constant pHout (= 7.3) ammonium (0-30 mmol/l) caused an increase of pHin from 5.5 to 6.6. The increase of pHin was accompanied by an increase of Km from 5 to 20 mumol/l 3H-catecholamine and by a decrease of both Vmax and delta p from 20 to 5 nmoles/(mg prot. X min) and from 123 to 85 mV, respectively. From the dependence of the Km of uptake on pHout is concluded that the uncharged species of catecholamine is transported, whereas the dependence of Km on pHin suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.
- [Show abstract] [Hide abstract] ABSTRACT: La tétrabénazine (TBZ) et la réserpine sont deux inhibiteurs du système de pompage des catécholamines de la membrane du granule chromaffine. Analogues structuraux de deux substrats, la dopamine et la sérotonine, ils agissent sur le transporteur des monoamines, qui catalyse l'échange H+/amine neutre. La dihydrotétrabénazine[3H] (TBZOH[3H]) se lie à la membrane du granule sur une classe de sites (sites T, KD = 3 nM); la réserpine[3H] se lie sur les sites T et sur une deuxième classe de sites (sites R1, KD = 0,7 nM). Les deux sites sont impliqués dans le transport. Les substrats déplacent les ligands avec des efficacités différentes: la noradrénaline (Km = 10 μM) déplace efficacement la réserpine (EC50 = 30 μM) et avec peu d'efficacité la TBZOH (EC50 = 2000 μM); la m-iodobenzylguanidine, un substrat récemment reconnu du système de transport des monoamines (Km = 5 μM) est efficace vis-à-vis de la TBZOH (EC50 = 25 μM) et inefficace vis-à-vis de la réserpine (EC50 = 300 μM). Les deux substrats utilisant le même transporteur, ce résultat confirme l'existence de deux sites aux propriétés différentes. Les sites T sont caractérisés par l'existence d'une relation linéaire entre l'inverse de la constante de dissociation des différentes drogues déplaçant la TBZOH[3H] et leur coefficient de partage en milieu octanol/H2O. Cette relation, qui indique la nature hydrophobe des sites T, n'existe pas pour les sites R1. Les sites T ont été identifiés par marquage covalent à l'aide d'un dérivé de la TBZ couplé à un groupe arylazido. Le site marqué est porté par une protéine d'environ 65 000 de masse moléculaire.
- [Show abstract] [Hide abstract] ABSTRACT: Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)