Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [³H]GBR 12783: Attempts to Characterize the Na⁺ Dependence of the Neuronal Transport of Dopamine

Abstract

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.

The Role of N-Glycosylation in Function and Surface Trafficking of the Human Dopamine Transporter

“…The present results did not indicate a preferential exclusion of partially or non-glycosylated DAT from the plasma membrane even though there was a tendency for less glycosylated material to be somewhat underrepresented in the surface fraction. In fact, the protein content of the surface fraction was reduced in the double and triple mutant compared with wild type to the same extent as that in the total fraction, consonant with the conclusion of Torres et (25)(26)(27). Taken together the data suggest that both non-glycosylated and mature DAT reaches the surface.…”

Multisubtrate mechanism for the inward transport of dopamine by the human dopamine transporter expressed in HEK cells and its inhibition by cocaine

“…Inward transport could be blocked with 100 µM cocaine, and is dependent on Na ϩ and Cl -with reaction orders of 2 and unity, respectively. These results are in agreement with previously published studies (Krueger, 1990;Amejdki-Chab et al, 1992a;Wheeler et al, 1993) on the uptake of [ 3 H]dopa- mine into striatal preparations as well as those studies where nonradiolabeled dopamine was monitored (McElvain and Schenk, 1992;Povlock and Schenk, 1997). In HEK hDAT cell suspensions, [Na ϩ ] or [Cl -] reduction decreases the V max and increases the K m for dopamine inward transport (Fig.…”

Binding of the [125I]3?-(Iodophenyl)tropan-2?-carboxylic acid isopropyl ester to the dopamine transporter at a physiologically relevant temperature: Mutually exclusive binding and different ionic requirements for various uptake blockers and substrates

“…On the other hand, increasing [NaCl] from 50 to 134 mM enhanced the ability of DAT substrates dopamine and amphetamine to displace [ 125 I]RTI-121. With a similar buffer system at 0°C, Bonnet et al (1990) reported that uptake blockers (mazindol, nomifensine, methylphenidate, and cocaine) and substrates (DA and amphetamine) were equipotent in their ability to compete with [ 3 H]GBR 12783 when [Na 1 ] was increased from its peak value, 10 mM, to 130 mM (Amejdki-Chab et al, 1992b;Benmansour et al, 1987). With A Ringer medium containing 140 mM NaCl at 4°C, Maurice et al (1991) noticed that the abilities of DA and amphetamine to displace [ 3 H]BTCP were greatly increased compared with a 50-mM phosphate buffer.…”