Chemical evidence that catecholamines are transported across the chromaffin granule membrane as noncationic species.

Abstract

Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process under conditions in which the substrate exists primarily as a positively charged or neutral species. In order to distinguish between these two states, we studied the transport properties of a permanently charged quaternary analogue of epinephrine, (R,S)-dimethylepinephrine. We found that this compound was a classical competitive inhibitor of (R)-[3H]norepinephrine uptake, with a K; of 3.8 mM for the racemic form, or 1.9 mM for the R form. However, the [3H]dimethylepinephrine was not transported at all into granules. Our control for steric hindrance as an explanation for deficient translocation was analysis of the transport properties of isoproterenol, a secondary catecholamine with an isopropyl group around the amine residue. (R)-Isoproterenol was an effective competitive inhibitor of (R)-[3H]norepinephrine transport, with a K; of 91 ,uM. In contrast to dimethylepinephrine, (RS)-[3H]isoproterenol was clearly translocated across the granule membrane, with a Km of 123 aiM, or 61.5 IAM for the R isomer. Thus, the positive charge on dimethylepinephrine and not the size of the amine moiety appeared to be responsible for the lack of translocation. We interpret these data to indicate that, although the positively charged species can interact with the transport site, an uncharged species is the one actually transported.Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process, which has become increasingly attractive as a general and simple system to analyze chemiosmotic energy coupling to transport (1-5). Many models have been proposed to explain the process, but distinguishing among them has been made difficult by lack of knowledge of whether the neutral, cationic, or anionic catecholamine species is the true substrate (4,(6)(7)(8)(9). Recently, on the basis of observations that both the binding constants for dopamine and serotonin and the calculated cationic fractions were constant between pH 6.8 and 7.6, Knoth et al. (10) concluded that the cationic species was translocated. However, using a similar experimental approach, Scherman and Henry (11) came to the opposite conclusion. They observed that the Km for norepinephrine declined between pH 6.5 and 8.5, and they interpreted this as due to an increase in the concentration of the uncharged species, the proper substrate. Yet, on the basis of entirely different considerations, Johnson and Scarpa (7) initially claimed that the species transported was most likely neutral, and more recently summarized by saying that a conclusion was not yet possible (8).We therefore decided to reinvestigate this question directly by examining the transport properties of a quaternary analogue of epinephrine. The compound we used was (R,S)-dimethylepinephrine (Me2E). * Me2E and its tritium-labeled derivative have the structure shown in Fig. 1 FIG. 1. Structures of Me2E (A) and isoproterenol (B). [3H]Me2E waslabeled at the p carbon (*). The wavy line on isoproterenol in ...

Uptake of norepinephrine and related catecholamines by cultured chromaffin cells: characterization of cocaine-sensitive and -insensitive plasma membrane transport sites.

“…Isoproterenol, however, is both a competitive inhibitor and a substrate (19). Thus, the positive charge and not the size of the bulky group around the amine moiety seemed to be responsible for the inability of dimethylepinephrine to be transported across the granule membrane.…”

“…As shown in Table 3, the cocaine-sensitive transport of (R)-norepinephrine was inhibited by a series of related compounds with varying efficiencies as defined by IC50. However, dopamine and epinephrine were selected for closer study because of their physiological importance, and isoproterenol and dimethylepinephrine were selected because of previous extensive studies on their transport through the chromaffm granule membrane (19).…”

“…In the chromaffin granule transport system, isoproterenol is a competitive inhibitor of norepinephrine transport (19) and a substrate for transport in its own right (19,34). In contrast, dimethylepinephrine, a permanently charged quaternary catecholamine, is a competitive inhibitor of norepinephrine transport but is itself not transported (19). In the case of transport across the chromaffin cell plasma membrane, however, both isoproter-Biochemistry: Banerjee et al enol and dimethylepinephrine were taken up by a cocaineinsensitive low-affinity uptake site ( Table 4).…”

“…Both the ATPase(s) (14,15) and the transport site(s) (16,17) have been purified and functionally reconstituted. At a more chemical level, the specificity of the uptake system for different catecholamine analogues has also been extensively studied by us as well as by others (18)(19)(20)(21).…”

Identification of Residues Involved in Substrate Recognition by a Vesicular Monoamine Transporter

“…At physiologic pH, monoamine substrates for VMAT1 and VMAT2 exist in both neutral and protonated forms. Previous studies had suggested that the transport protein recognizes the neutral form (Ramu et al, 1983). However, more recent work has shown the vesicular transport of permanently charged substrates such as MPP ϩ (Scherman et al, 1988;Daniels and Reinhard, 1988;Moriyama et al, 1993), strongly suggesting that the transporter recognizes the protonated form of monoamine substrates (Schuldiner et al, 1995).…”