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Comparison of DABA and GABA Transport Into Plasma Membrane Vesicles Derived from Synaptosomes

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Comparison of DABA and GABA Transport Into Plasma Membrane Vesicles Derived from Synaptosomes Jourtiol of Neurochemisiry 0022-3042/8 110201 -05441S02.0010 36(2):544-550. February. Raven Press, New York Q 1981 International Society for Neurochemistry Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes Robert Roskoski, Jr. Depurrrnrnr of Biocliemistrj. Louisiana State University Medicrtl Center. Neil, Orleans, Louisiunu 701 12, U.S.A Abstract: Transport of GABA by a high-affinity transport system (K, = M) is thought to terminate the action of this postulated neurotransmitter. 2,4- Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [:1H]DABA by auto- radiography has been used to identify neurons with the GABA high-affinity trans- port system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive ['HJGABA and ['HIDABA trans- port. For each. transport requires external sodium, is abolished by ionophores that dissipate the Na' gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor (K, = 0.6 mM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experi- ments, using enzymatic-fluorometric techniques, it was shown that the artifi- cially imposed ion gradients drive net chemical transport of GABA into the vesicles. Key Words: GABA-DABA-Synaptosomes-Membrane vesicles- Transport. Roskoski R., Jr. Comparison of DABA and GABA transport into plasma membrane vesicles derived from synaptosomes. J. Neurochern. 36, 544-550 (1981). GABA (y-aminobutyric acid) is a postulated neu- neurotransmitter action by uptake (Iversen, 1971). rotransmitter in the vertebrate CNS (Krnjevic, Levi and Raiteri (1974) and Simon et al. (1974) re- 1970). Nerve terminal preparations (synaptosomes) ported that the high-affinity synaptosomal transport accumulate GABA by high-affinity (low K,) and system mediates the exchange of endogenous and low-affinity (high K,) transport systems (Weinstein external GABA, but little net transport. We demon- et al., 1965; Martin, 1973; Levi and Raiteri, 1974). strated that synaptosomes take up net amounts of Two similar classes of transport systems occur for GABA, glutamate, and aspartate by the high- other neuroactive amino acids (Iversen and John- affinity system (Ryan and Roskoski, 1977; Ros- ston, 1971; Logan and Snyder, 1972). The low-af- koski, 197th; 1979); exchange of each amino acid finity transport systems are associated with both also occurs. We suggested that high-affinity trans- neuroactive and inactive amino acids (Logan and port is reversible and that the direction of movement Snyder, 1972). The high-affinity systems, which are may be related to the bioenergetics and mechanism Na+- and temperature-dependent are associated of uptake. with the neuroactive amino acids, including GABA. Iversen and Johnston (1971) demonstrated that It has been suggested that the high-affinity systems 0.1 mM-DABA (2,4-diaminobutyric acid) inhibits are specifically involved with the termination of [3H]GABA accumulation in brain slices. Simon and Received January 30, 1980; accepted July 25. 1980. 2.4-Diaminobutyric acid: Mes. 2-(N-Morpholino) ethanesul- Abbreviations used: GABA. y-Arninobutyric acid; DABA, fonic acid. 544 DABA AND GABA TRANSPORT INTO PLASMA MEMBRANE VESICLES 545 Martin (1973) studied the transport of both GABA with 2 ml of 0.20 M-NaCI. After drying (5 min. IIO"), and DABA into rat brain synaptosomes. They radioactivity was measured by liquid scintillation spec- showed that DABA is a competitive inhibitor of trometry, using Budget-Solve (Research Products Inter- 13H]GABA uptake. Using low-specific-activity national Corp.) as scintillant with an efficiency of 35%. To measure background radioactivity. the ice-cold 0.2 hi- ["CIDABA, they showed that its uptake was par- NaCl was added before the membrane fraction; filtration tially inhibited by GABA. They pointed out that and washing were then performed immediately. as de- there may be a population of synaptosomes active scribed. in DABA, but not GABA, uptake. Kelly and Dick Each measurement was performed in duplicate: the (1975) reported that 13H]DABA is taken up by agreement was within 1057, or the measurement was re- neuronal elements, but not in glia in rat cerebellum. peated. There is a two- to threefold variation in rates and These investigators emphasized the similarity of extent from one membrane preparation to another based distribution of [3H]DABA and ["IGABA. on protein. Values from a given membrane preparation, Several investigators have prepared membrane however, varied only 10-15%. Each experiment was vesicles which take up substrates in response to ar- performed with at least three membrane preparations and tificially imposed ion gradients. Kanner (1978), for similar results were obtained. example, prepared fractions from synaptosomal membranes active in GABA transport. In the pres- Transport Measurement in Synaptosomes ent study the characteristics of [3H]DABA and [3H]GABA transport into these vesicles were com- These experiments were performed by the method pre- pared. Although there are many similarities, sub- viously outlined for choline uptake (Roskoski, 19786). stantive differences exist, which raise the possibil- Transport was terminated by addition of 4 ml of ice-cold 0.9% NaCl followed by filtration through GFiA filters; the ity that DABA is transported, at least in part, by a filters were washed twice with two additional 4-ml por- non-GABA transport system. tions. MATERIALS AND METHODS Marerials Preparation Membrane Fractions of Labeled GABA and DABA were obtained from New Rat cortical synaptosomes were prepared from male England Nuclear Corp. and Amersham. respectively. Sprague-Dawley rats (125-200 g) by the method of Nigericin and monensin were gifts from Eli Lilly and Co. Haycock et al. (1978). The synaptosomes were lysed and Other drugs and compounds were purchased from Sigma the plasma membrane fraction was prepared by the pro- Chemical Co. cedure of Kanner (1978). The lysed membrane fraction was stored in 200-pl aliquots [about 5 mgiml protein de- termined by the procedure of Lowry et al. (1951)l in RESULTS liquid N,. General Characteristics of DABA and GABA Transport Transport Assays Using 0.14 FM labeled substrate, the rates of Transport measurements were carried out by the meth- DABA and GABA transport into reconstituted ods of Rudnick (1977) and Kanner (1978). with minor vesicles are linear for about 30 s (Fig. 1). GABA modifications. Unless otherwise noted, the thawed mem- uptake exhibits a characteristic overshoot. With 1.O brane fraction was reconstituted in 4 volumes of 100 mM substrate under the same experimental condi- mwKCI. 10 mwNaCI, and 90 mM-choline chloride tions, there is appreciable DABA uptake (Fig. 1). buffered with 5 mal-Mes-Tris (pH 7.0) at a concentration Maximal uptake of DABA occurs by 1 and 4 min, of about 1 mgiml protein for 10 min at 37". After centrifu- gation (27.000 x gfor 10 min). the membranes were resus- respectively, with the low and high substrate con- pended (about 1 mgiml protein) in fresh solution of the centrations. Although GABA transport can be same composition by vortex mixing. To measure trans- demonstrated with higher protein concentrations, it port, a 20-pl portion was transferred to 180 p1 of transport is not readily detected under these conditions with a solution consisting of 100 mwNaCI plus 100 mM-choline 1 .O mM concentration because of the relatively low chloride buffered with 5 mwMes-Tris (pH 7.0). This pro- specific radioactivity. The time course of transport cedure generates Na* (Naa,,u,c,r,,> Na+lnsldr)and K' of GABA into synaptosomes differs from that into (K- ,",,,,,, > K',,, ,,,, r,r) gradients. The external transport so- vesicles (Fig. 1). Uptake plateaus after 10 min in lution also contained ["HIGABA (26,600 c.p.m.ipmol) or synaptosomes and after 2 min in the membrane L-["H]DABA (6850 c.p.m.ipmol) to give a final concen- vesicles. The former, but not the latter, possesses tration of 0.14 pxi. After incubation for the specified time at ambient temperature (22-24"), transport was termi- the metabolic machinery to maintain ion gradients. nated by addition of 2 ml of ice-cold 0.20 M-NaCI in 5 Dissipation of the artificially imposed ion gradients mxi-Mes-Tris (pH 7.0) and filtration through 25-mm- occurs rather rapidly. diameter Whatman GFiA glass-fiber discs. The filtration Experiments were next performed to determine rate was 2 ml per 2-4 s. The filters were washed twice the ionic requirements for transport. Both DABA J. Neiirochem.. Vd.36. No. 2, 1981 546 R. ROSKOSKI 8 5 10 15 lime, min Time, min FIG. 1. Time course of DABA and GABA uptake. Trans- port into membrane vesicles and synaptosomes was mea- sured by procedures given in Materials and Methods. (A) GABA (A-A) and DABA (0-0)transport into membrane vesicles using 0.14 ~LMlabeled substrate concentrations. (B) DABA transport into membrane vesicles using a 1.0 mM concentration. (C) GABA (0-0)and DABA (0-0) transport into synaptosomes using 0.1 4 p~ radioactive substrate. The values given are the mean of duplicate de- terminations. Similar results were obtained with four dif- ferent preparations. lime, min and GABA transport are Na+-dependent (Table 1). concentration gradient (from inside to outside) Substitution of Li+ or choline for Na+ decreased valinomycin also produces a more negative intra- apparent uptake by 98%.
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