Coupling of transmembrane proton gradients to platelet serotonin transport.

A pH difference (acid inside) across the platelet plasma membrane increases both the rate and extent of serotonin accumulation inside plasma membrane vesicles. Even in the absence of other transmembrane ion gradients, this pH difference (delta pH) serves as the sole driving force for serotonin accumulation, leading to a serotonin concentration 18-fold higher inside the vesicle. This process requires Na+ and is blocked by imipramine, indicating that it is mediated by the serotonin transporter. At physiological pH, internal K+ is counter-transported with serotonin, and high internal K+ stimulates transport maximally. Internal K+ also blocks the delta pH stimulation of serotonin transport. Conversely, low internal pH (5.6) inhibits the ability of internal K+ to stimulate transport. This apparent competition between K+ and protons suggests that delta pH drives serotonin accumulation through counter-transport with protons, and that serotonin is transported in its cationic form.

9 Established Investigator of the American Heart Association. resent activation of the Na+, K'-ATPase (Paton, 1976;Horn, 1976). Our results suggest that possibly also in these cases, neurotransmitter re-uptake is coupled to proton countertransport.

Methods
Preparation and Equilibration of Membrane Vesicles-The isolation of platelet plasma membrane vesicles from fresh porcine blood was performed as described previously (Barber and Jamieson, 1970;Rudnick and Nelson, 1978). Prior to assay, vesicles were diluted 20fold into a medium of 85 mM lithium or potassium gluconate, containing 1 mM MgSO,, approximately 10 mM N-(2-acetamido)iminodiacetic acid adjusted to pH 5.6, 6.5, or 7.5 with either KOH or LiOH, and 1 to 8 mM mannitol to keep the osmolarity constant at 200 mOSM. Buffer concentrations were adjusted so that in every case, the alkali cation concentration was 0.1 eq/liter. Lithium ion does not substitute for, or compete with, internal K' or external Na' (Rudnick, 1977;Nelson and Rudnick, 1979) and is used here as an inert cation. The suspensions were incubated at 37 "C for 15 min, sedimented at 48,000 X g for 20 min at 4 "C, and resuspended to the original volume in the incubation buffer.
Transport Assay-Serotonin transport was assayed at 25 "C by 40fold dilution of a pre-equilibrated vesicle suspension (20 pg of membrane protein) into 92 mM NaCl, containing 1 mM MgS04, 0.06 PM 5hydroxy[l,2-"H]tryptamine (17,000 cpm/pmol), approximately 5 mM ADA' adjusted to pH 5.6, 6.5, or 7.5 with NaOH, and adjusted to 200 mOSM with 0 to 4 mM mannitol. Again, the Na' concentrations was always 0.1 eq/liter. Initial rates of transport were measured only during the first 15 s. Reactions were stopped by filtering the vesicle suspensions on nitrocellulose filters (Millipore HAWP), washing with cold 0.1 M NaCl, and counting as described previously (Rudnick, 1977).
Efflux Measurements-Vesicles (80 pg of membrane protein) equilibrated as described above with lithium gluconate buffer at pH 5.6 or 7.5, were diluted 10-fold into NaCl buffer at the same pH, containing 30 to 160 nM ['Hlserotonin. After 2 min at 25 "C, the reaction mixture was diluted 40-fold with NaCl medium of the pH indicated in the figure legend, containing 1 p~ imipramine. The incubation was continued at 25 "C and 0.5-ml aliquots were taken at 5 s, and 1, 2, 3 min. Efflux was terminated by filtration as described previously . The loss of accumulated 5-hydroxytryptamine was linear with time under these conditions. Protein Determinations-Protein was determined by the method of Lowry et al. (1951). serves as a driving force for serotonin accumulation by platelet plasma membrane vesicles. The experimental results shown in Fig. 1 demonstrate that in the absence of a transmembrane Na' gradient, ApH drives the carrier-mediated transport of serotonin. Membrane vesicles equilibrated with NaCl medium buffered at pH 5.6 and diluted into the same medium buffered at pH 7.5 accumulate serotonin to a significantly higher level than if no ApH is imposed. From the internal volume of the vesicles (12 pl/mg of protein, Rudnick, 1977), we calculated that the internal serotonin concentration was 18-fold higher than that of the medium. In this case, the only possible driving force for transport is the imposed ApH. The data in Fig. 1 represent that portion of the serotonin accumulation which is sensitive to the ionophore monensin and therefore dependent on ApH. This ionophore (which catalyzes electrically neutral exchange of protons and alkali cations, and is expected to dissipate both ApH and any residual Na' gradient) has no effect in the absence of a ApH at pH 5.6, 6.5, or 7.5, but dramatically inhibits ApH-driven transport.
Serotonin transport may be driven by ApH via many possible mechanisms. One possibility is that serotonin, a weakly basic amine, equilibrates across the membrane by nonionic diffusion of the more lipophilic free base, and is trapped inside by the high proton concentration (Nichols and Deamer, 1976). This possibility is unlikely to account for our results, however, since imipramine, a specific inhibitor of the serotonin transporter, completely blocks ApH-driven transport (Fig. 1). Moreover, little ApH-driven accumulation was observed when Na' was replaced by Li' and no transport in the absence of Na' was imipramine-sensitive (data not shown). Another possible explanation for ApH-driven transport is that the plasma membrane vesicle preparation is contaminated with small amounts of membrane vesicles derived from platelet storage granules, which are believed to accumulate serotonin in response to an ATP-generated ApH (Rudnick et aE., 1980). This possibility is unlikely for two reasons. First, the granular serotonin transport system is not Na'-dependent, and second, the serotonin accumulation shown in Fig. 1 is insensitive to reserpine, a specific inhibitor of the granule system (data not shown).
p H Rate Profile for Serotonin Transport-To define further the mechanism by which a ApH drives the serotonin transport system, we examined the effect of pH on the rate of transport. Fig. 2 shows the pH rate profiie for serotonin accumulation by vesicles equilibrated with either K' or Li'. We have previously shown that internal K' increases the rate and extent of serotonin accumulation (Rudnick, 1977;Rudnick and Nelson, 1978;Nelson and Rudnick, 1979). Therefore, although we expected faster transport into K'-loaded vesicles, we were surprised by the different shape of the pH rate profiles. While transport into Kc-containing vesicles rises to a maximum rate between pH 6.5 and 7.5, and does not decrease in rate at high pH, vesicles lacking K+ show a distinct pH optimum at approximately pH 6.5, and transport more slowly at high pH. Furthermore, the K' stimulation (relative to Li') varies over the pH range tested, from a 5-to &fold stimulation at pH 7.5 to essentially no difference at pH 5.6.
Time Course of Serotonin Transport-The time course of serotonin transport reflects not only the influence of both ApH and external pH, but also additional factors. Fig. 3 demonstrates that when plasma membrane vesicles isolated from platelets are equilibrated in Na'-and K'-free buffer at pH 5.6 and then diluted into NaCl medium buffered at pH 7.5, serotonin accumulates within the vesicles to a much higher extent than if no ApH is imposed at either pH 5.6 or 7.5. Immediately after dilution into Na' medium, the vesicles accumulate serotonin rapidly and reach a peak serotonin NaCl containing 1 mM MgS04, 5.3 to 9.2 mM ADA adjusted to pH 5.6, 6.5, or 7.5 with NaOH (the final Na' concentration was always 0.1 eq/liter) and 0.8 to 4.7 mM mannitol to adjust the osmolarity to 200 mOSM. These Na'-loaded vesicles were diluted into the same medium adjusted to pH 5.6, 6.5, or 7.5, containing 0.06 p~ ['Hlserotonin. Monensin-treated controls (5 p~) were subtracted from each of the time points. The same amount of serotonin was accumulated in the presence of monensin or imipramine, a maximum of approximately 20 pmol mg". W, pH 5.6 in = out; M , pH 5.6 in, pH 7.5 out; H , pH 6.5 in = out; [1"-I7, pH 7.5 in = out; A-A, pH 5.6 in, pH 7.5 out + 1 pM imipramine. content at 5 to 10 min after dilution. As the ion gradients which drive serotonin transport decay with time, the accumulated serotonin gradient also decays, until little intravesicular serotonin remains 2 h after dilution. This result suggests that ApH acts an an additional driving force for serotonin transport even when superimposed over pre-existing transmembrane gradients of Na' and C1-. The data presented in Fig. 4 indicate that the maximum extent of serotonin accumulation by K+-loaded vesicles also is much higher when a ApH (5.6 in, 7.5 out) is imposed across the membrane than at pH 5.6 or 7.5 in the absence of ApH.
However, Figs. 3 and 4 also show the time course of transport into vesicles equilibrated and diluted with media at an intermediate pH, 6.5. In the absence of internal K' (Fig. 3 ) , serotonin influx is slower at pH 6.5 (in = out) than with a ApH, in contrast to the results in Fig. 2 which indicate faster transport at pH 6.5 than at 5.6 or 7.5. The only difference is that in the experiment shown in Fig. 3, a ApH was imposed (the internal pH was 5.6 instead of 7.5). The effect of internal Time course of serotonin accumulation by platelet plasma membrane vesicles. Vesicles were equilibrated with lithium buffer and assayed in buffered NaCl as described under "Experimental Procedure," stopping the reactions at the indicated times. Filled symbols, internal pH = 5.6. Circles, external pH = 5.6. Squares, external pH = 7.5. Triangles, pH 6.5 in = out. pH on transport rate will be described below. In the absence of a ApH, at pH 6.5, the vesicles transport serotonin to almost the same level (Fig. 3) or even higher IeveIs (Fig. 4 ) than when ApH is imposed, in contrast to the results shown in Fig. 1, in the absence of an Na+ gradient.
It is clear from Figs. 3 and 4 that the peak level of serotonin accumulated within the vesicles reflects not only the forces driving serotonin across the membrane, and the rate at which serotonin equilibrates with these driving forces but also the rate at which these forces (the transmembrane ion gradients) decay. For example, at pH 7.5, with no ApH, serotonin enters the vesicle rapidly, but fails to reach high internal concentrations, possibly because the ion gradients decay too fast. As another example, in the presence of internal K' at pH 6.5, the serotonin gradient is maintained much longer than when a ApH is imposed, suggesting that the ion gradients are more stable under these conditions. It is clear, therefore, that imposition of a ApH on top of pre-existing ion gradients affects many aspects of transport, some of which may obscure any coupling between proton and serotonin transport. Although it is customary to use transport time courses such as shown in Figs. 3 and 4 as evidence for coupling between substrate and proton transport, our experience suggests that this approach may give misleading results.
Stimulation of Transport Rate by Low Internal pH-Internal K+ is thought to stimulate serotonin transport by facilitating interconversion of the transporter from the form which releases internal serotonin to a form which can bind external serotonin. Potassium ion leaves the vesicle in the process . To test the possibility that a low internal pH acts in the same way, we measured the effect of ApH on the initial rate of serotonin transport into vesicles equilibrated with either K' or Li+. The results, shown in Table I, demonstrate that at high external pH (7.5) either a low internal pH or high internal Kf stimulates the rate of transport. With K+-loaded vesicles at pH 5.6 (in = out), the rate is slow, as also shown in Fig. 4. If the internal pH is raised to 7.5, the rate is also low, suggesting that low external pH (5.6) is sufficient to slow down transport. If the external pH is raised to 7.5 (pH 5.6 inside), however, the rate increases dramatically. At pH 7.5 outside, the rate is still controlled primarily by the external pH, since raising the internal pH from 5.6 to 7.5 has no effect.
In contrast, when internal K+ is replaced with Li+, both internal and external pH determine transport rate. As with K+-loaded vesicles, increasing internal pH has little effect (external pH = 5.6), and increasing external pH dramatically increases the rate (internal pH = 5.6). In the absence of

Stimulation of transport rate by L\pH in the presence and absence of internal K'
Initial rates of transport (15 s) were measured as described under "Experimental Procedures" using the conditions given in the table.
Results are reDorted as aicomoles min" me" f S.D. internal K', however, the maximal transport rate requires an acidic interior. If the internal pH is raised to 7.5, transport slows down to the rate in acid media. Moreover, the transport rate in the presence of a ApH (interior acid) is faster than at the pH optimum (pH 6.5 in = out), and approximately twothirds of the rate with K'-loaded vesicles under the same conditions. One possible mechanism by which an acidic interior might stimulate transport is by decreasing the fraction of internal serotonin in the neutral (free base) form, thereby decreasing passive efflux. To test this possibility, we measured rates of passive efflux under various conditions. Vesicles were actively loaded to various internal serotonin concentrations at various pH, and diluted into NaCl medium containing imipramine at a concentration which completely blocks efflux via the serotonin transport system (Talvenheimo et al., 1979). The results in Fig. 5 show the dependence of passive efflux upon internal and external pH. As expected, efflux is slowest at low pH and fastest at high pH (in = out), and does not appear to saturate as the internal serotonin concentration is increased. However, the stimulation of transport rate by low internal pH (Table I) is much faster than this passive efflux. Even under conditions where passive efflux is fastest, it accounts for less than 7% of the increase in net influx due to a ApH. Thus, stimulation by low internal pH is due to an increase in influx, and not a decrease in passive efflux.
Competition between H+ and K+-If internal protons substitute for internal K', stimulation by K+ should vary with internal pH. This phenomenon is demonstrated by the data shown in Fig. 6. In this experiment, we measured the initial rate of serotonin transport into K+-loaded and K+-free vesicles as a function of internal pH, and plotted the ratio of the two rates. In one set of points (filled circles) the external pH varied with internal pH, but the open circles represent rates where the external pH was held constant at 7.5. In both cases, Rates of efflux were determined as a function of internal serotonin concentration as described under "Experimental Procedures" using vesicles equilibrated and actively loaded at the internal pH given below, and diluted into imipramine-containing NaCl medium at the indicated external pH. Open circles, pH 5.6 in, 7.5 out; filled circles, pH 5.6 in = out; open squares, pH 7.5 in = out; filled squares, pH 7.5 in, 5.6 out.

FIG.
6. K+ stimulation of transport rate as a function of internal pH. Initial rates of transport (15 s) were measured as described under "Experimental Procedures" using vesicles equilibrated in K' or Li' media buffered at the indicated pH. Vesicles were assayed at either the same pH as the equilibration medium (filled circles) or at pH 7.5 (open circles). The data represent the rates obtained with K'-loaded vesicles divided by the rates obtained with Li'-loaded vesicles.
the stimulation by K' increases with pH. At pH 5.6, K' stimulates only slightly. Even when a ApH is imposed by raising external pH, K' fails to markedly increase the rate, although both rates are at least 4-fold faster at high external pH. As the internal proton concentration decreases, internal K' stimulates more, 3-fold more at pH 6.7 as reported previously (Nelson and Rudnick, 19791, and 5-to 6-fold more at pH 7.5. External pH has little effect on this stimulation. Internal K' stimulates by the same factor whether the external pH is 7.5 or equal to the internal pH. Thus, internal protons appear to compete with K+ in accelerating serotonin transport.

DISCUSSION
There are many possible mechanisms by which a pH difference (ApH) across the platelet plasma membrane might stimulate the rate and extent of serotonin accumulation. The simplest possibility is that by influencing the distribution between the protonated, cationic form of serotonin and the more permeant, neutral form, the ApH traps serotonin on the acidic side of the vesicle membrane. For example, in liposomes, serotonin and dopamine are concentrated in response to ApH in the absence of other driving forces or transporters (Nichols and Deamer, 1976). Such a simple mechanism can not account for our observation that ApH-driven transport in platelet membrane vesicles is blocked by imipramine and requires Na'. Obviously, the ApH acts through the serotonin transporter.
A more interesting possibility is that the neutral form of serotonin is the true substrate, rather than the cationic form which predominates at physiological pH. Thus, a ApH might stimulate the rate and extent of transport by increasing the concentration of substrate on the outside and decreasing it on the inside. In most cases it is impossible to distinguish this type of stimulation from one where protons are counter-transported out of the vesicle as amine molecules are transported in. For example, in the chromaffin granule it has been impossible to distinguish between exchange of 1 proton with 1 neutral amine molecule on one hand and exchange of 2 protons for a protonated amine molecule on the other hand (Kanner et al., 1980;Knoth et al., 1980;Johnson et al., 1981). Knoth et al. (1981) recently proposed that in chromaffin granules only the cationic form of the substrate is transported, but their conclusions are based on many assumptions which have not yet been tested.
Fortunately, in the case of the serotonin transporter, the unique antagonism of ApH by internal K' provides a way to distinguish between these two mechanisms. If ApH acted merely to change the substrate concentration, there would be no interaction between internal protons and internal K'. Yet, it is clear that high internal K+ blocks the stimulation by internal protons while high internal proton concentrations inhibit stimulation by internal K' (Table I). In light of the proposal that K' and serotonin are counter-transported, the simplest interpretation of our data suggests that when K+ is not present internally, protons replace K+ and are countertransported with serotonin. A second argument in favor of counter transport is that internal protons stimulate the initial rate of transport under conditions where serotonin efflux is negligible. Fig. 7 presents this interpretation in schematic form. After the transporter releases serotonin, Na', and C1-on the vesicle interior (Kd), it binds either K' or H', and returns to an http://www.jbc.org/ Downloaded from "external" form (either T,K' or T,H+), which releases K' or H' to the external medium, forming To. This form of the transporter binds external Na', serotonin, and C1-( k J , and transports them to the interior (kin). In the absence of internal K' , the rate of this cvcle is determined by the internal pH. At physiological pH or higher, where T,H+ is low, internal K' stimulates transport markedly by providing another pathway (kout(K+)). At low internal pH, where protons are not as limiting, K+ only slightly increases the rate. This mechanism easily explains the pH rate profiles shown in Fig. 2. Just as external K' inhibits transport , low external pH limits transport, regardless of internal K' . Up to pH 6.5, the rate of transport increases with pH. At this point, the rate becomes limited by internal pH (TiH+), and decreases as the internal proton concentration drops. When internal K' is present, however, it can fulfil the internal cation requirement and the rate remains high at high pH.
The substitution of HC for KC dictates that the same number of charges will cross the membrane in K'-loaded or K'free vesicles. Thus, if the transport system is electroneutral when internal Kt is present (Rudnick and Nelson, 1978;Nelson and Rudnick, 1979), it should also be electroneutral in the absence of K' . The glutamate transport system of mammalian kidney also is stimulated by, but does not require, internal K+ , and is electroneutral in the presence or absence of internal K' . Our results suggest that also in this transport system H' may replace K' . Supporting this suggestion is the observation that (as with serotonin transport), etimulation of glutamate transport by K' is diminished at low pH . Another glutamate transporter in rat brain is thought to absolutely require internal K' for activity (Kanner and Sharon, 1978). An intriguing possibility is that the kidney and brain transporters are identical, but that in its microenvironment in the brain, the transporter pK, decreases to the point where internal protons cannot replace K' at neutral pH. The system would therefore appear like the serotonin system at high pH, where internal K' is almost essential.
Given that the intracellular K' concentration is high, the physiological role of ApH-driven serotonin transport may be negligible. However, stimulation by ApH represents another distinctive feature of this system which can be tested with other neurotransmitter transporters, such as those for glutamate, norepinephrine, and dopamine. Furthermore, our results indicating that ApH does not stimulate in the presence of internal K' render unlikely the possibility that serotonin is transported in its neutral form, and further define the stoichiometry of the transporter.