Correspondence to: Chin-Chih Lu, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75325-9040. Fax:Fax: 214-648-8879; E-mail:chinchih{at}iname.com or hilgeman@utsw.swmed.edu.
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Abstract |
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Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent -aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl-- dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, currentvoltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.150.3 equivalent charges. The slopes of currentvoltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.
Key Words: neurotransmitter transporter, NO-711, patch clamp, substrate translocation, voltage dependence
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Introduction |
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Neurotransmitter transporters, many of which are of great medical importance (-aminobutyric acid (GABA),1 glycine, serotonin, and catecholamines and the family of excitatory amino acid transporters (
Stoichiometric transport of substrates is usually explained by the "alternating access" hypothesis (
To address these issues, we have characterized GAT1 function with the Xenopus oocyte expression system, using giant membrane patches to record GAT1 currents (
The reaction cartoons, shown in Figure 1, are intended to orient the reader to our overall goal; namely, to account for the dependencies of GAT1 function on all three substrates on both membrane sides, to account for voltage dependencies of transport, and to account for transporter kinetics. Figure 1 A depicts groups of reactions thought to occur in the forward transport mode of GAT1 (i.e., moving GABA into cells) on the basis of previous work. Perhaps the most important detail is that a slow reaction can occlude one Na+ ion from the extracellular side in the absence of GABA and chloride ( = 15200 ms), and it probably rate-limits inward current in the middle potential range, where the slope of the currentvoltage relations of the transport current is quite steep. Evidently, GABA and chloride can bind only after Na+ has been occluded by the transporter, and when they do so, all three substrates are transported to the cytoplasmic side via a faster reaction (Figure 1, fast) that is nearly electroneutral. At large negative potentials where the "slow" charge-moving reaction is accelerated, this "fast" translocation reaction probably becomes rate limiting; this would explain the observed current saturation with hyperpolarization (
< 1 ms) can occur in GAT1 in the absence of substrates on both membrane sides (
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The results described in this article for the reverse transport mode (i.e., GABA transport from inside to outside) give rise to the reaction perspectives summarized in Figure 1 B. In the absence of extracellular substrates, the reverse transport cycle appears to be rate limited by a weakly voltage-dependent reaction under most conditions. According to our analysis, the underlying reaction must involve the occlusion and/or translocation of substrates from the cytoplasmic side. One possible scheme is shown in Figure 1 B, where one Cl- and one Na+ are occluded in a slow step, and all other reactions in the reverse cycle are much faster. These include (a) the binding of a second cytoplasmic Na+ and GABA, (b) electroneutral substrate transport and release to the extracellular side (Figure 1 B, fast), (c) the electrogenic deocclusion of Na+ (fast, with a large lightning symbol), and (d) the reorientation of empty binding sites that allows cytoplasmic Cl- and Na+ to bind again. We point out that the results presented in this article alone do not justify these interpretations, and that the interpretations alone do not allow formulation of a reaction scheme for GAT1. Thus, in an accompanying article (
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Materials and Methods |
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Transporter Expression
GAT1 cRNA was in vitro transcribed from pBSAMVGAT1 (gift of S. Mager, California Institute of Technology, Pasadena, CA) with T7 polymerase and injected into Xenopus oocytes, which were isolated and maintained according to
Electrophysiology
Giant inside-out oocyte membrane patches (612 pF; seal resistance > 1 G) were obtained as previously described (
Data Presentation
Many experiments described in this and the next article (
Currentvoltage relations for the outward GAT1 currents, presented in this article, are described well by a simple exponential (Boltzmann) function of the form, A · eq · 0.5 · Em · F/RT, where A is a scalar, Em is the membrane potential, F/RT has its usual meaning, and q is the equivalent charge. According to Eyring rate theory, and assuming an energy barrier midway through the membrane electrical field, the "equivalent charge" is the amount of charge that would have to move through the entire membrane field to account for the voltage dependence. This is "equivalent" to a proportionally larger charge amount that would move through a proportionally smaller fraction of membrane field to account for the same voltage dependence. RT/F was approximated as 26.5 mV.
Solutions
The standard bath solution contained (mM): 020 GABA, 120 NaCl, 0.5 magnesium sulfamate, 20 tetraethylammonium-OH, 10 EGTA, and 20 HEPES, pH 7.0. Unless noted otherwise, the standard pipette solution contained (mM): 20 N-methylglucamine (NMG)1-Cl, 100 NMG2-(N-morpholino)ethanesulfonic acid (MES), 2 magnesium sulfamate, 4 calcium sulfamate, 0.02 ouabain, and 20 HEPES, pH 7.0. Equimolar NMG was substituted for Na+, and MES was substituted for Cl-.
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Results |
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Basic Properties of GAT1-mediated Currents in Giant Oocyte Membrane Patches
Experimental definition of GAT1 current.
Giant membrane patches from control oocytes exhibited no GABA-activated currents (data not shown). Figure 2 A shows the typical GABA-activated currents recorded in giant membrane patches from oocytes expressing GAT1. Outward current is activated when GABA (20 mM) is added to a cytoplasmic solution containing 120 mM NaCl, whereby the extracellular (pipette) solution contains 20 mM Cl- and no GABA or Na+. Inward current is activated in the absence of cytoplasmic GABA, Na+, and Cl- when GABA (0.2 mM) is added via pipette perfusion with a pipette solution that contains 100 mM NaCl. Inward currents at 0 mV were typically manyfold smaller than outward currents at 0 mV. In stable patches, the GABA-induced currents showed no change in magnitude for over 30 min.
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Figure 2 B illustrates our standard protocol for studying the voltage dependence of steady state transport current. When the membrane current reached a new steady value after solution changes, the voltage protocol described in Figure 2 B (middle) was applied. This is the cause of the current spikes ad in Figure 2 A. Typically, we used cumulative membrane voltage steps of 30 mV magnitude from 0 to 150 mV, up to +90 mV, and back to 0 mV. The step durations were just long enough (17.5 ms in Figure 2 B) so that presteady state transients of the inward GAT1 current decayed for the most part during each step. Figure 2 B (top) shows the membrane current response during (a) and after (b) the application of cytoplasmic GABA, and the difference (ab) is shown below. The same procedure was used to obtain membrane current responses before (c) and during (d) extracellular GABA application by pipette perfusion. The subtracted record (dc) reveals presteady state current transients that are faster at negative potentials. To obtain the steady state currentvoltage (IV) relation of the subtracted current, the median current magnitude of the last 3 ms of each voltage step is plotted against membrane voltage (Figure 2 C). The IV relations are monotonic. There is little or no hysteresis in the outward IV relation, and the moderate hysteresis present in the inward IV relation is expected from the slow time-dependent processes in the forward transport cycle (
Figure 3 shows that outward transport currents can be defined by two different means with very similar results. The extracellular solution contains Na+ (120 mM) but no GABA to test for GAT1-mediated inward current in the absence of GABA. (Figure 3, ) Definition of transport current by applying and removing 20 mM GABA from the cytoplasmic side; (
) the definition by applying NO-711, a high-affinity GABA uptake inhibitor, from the cytoplasmic side in the presence of 20 mM cytoplasmic GABA. The results are fitted by the Boltzmann equation, given in MATERIALS AND METHODS, and the equivalent charge is 0.63. This slope is two-times larger than in the absence of Na+o. (Figure 2 C) Pipette perfusion experiments that define the effect of Na+o in IV's in a single experiment are described in Figure 6 A of a companion article (
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Failure to detect GABA-independent GAT1 currents.
In re-lated experiments, we used NO-711 to further test whether GAT1 mediates ionic currents in the absence of GABA. In patches expressing GAT1, current responses to membrane voltage changes before and after applying NO-711 were compared, and no NO-711inhibited current was clearly detected. Protocols used to define GAT1 charge movements (
Outward IV relations were also defined by addition and removal of cytoplasmic Cl- or Na+, leaving the other substrate concentrations constant (not shown). Results for Cl-, using MES as the Cl- substitute, were virtually identical to those with GABA subtraction. This indicates that, in the absence of cytoplasmic Ca2+, the Cl- conductance of the oocyte membrane patches is very small. Results for Na+, using NMG as the Na+ substitute, were also very similar to those with GABA subtraction, provided that GAT1 expression was high and the endogenous Na+ conductance of the oocyte patch had run down. In general, however, the Na+ conductance of the oocyte membrane complicates results obtained with Na+ subtraction.
Strict cosubstrate requirements of outward GAT1 current.
In our experience, activation of outward GAT1 transport current in oocyte patches requires the simultaneous presence of GABA, Na+, and Cl- on the cytoplasmic side. Activation by each substrate in the presence of cosubstrates is demonstrated in Figure 4. The results were obtained using a fast solution switch device whereby the pipette tip is moved in <3 ms through the interface between two solution streams by a computer-controlled manipulator (
Figure 4 B shows the record obtained on applying 120 mM Cl-i, instead of 120 mM MES-i, in the presence of 20 mM GABAi and 120 mM Na+i. Current activation is just as fast as with GABAi because the half-maximal Cl-i concentration is only ~10 mM at 0 mV (see Figure 8 F). The current relaxation on removal of Cl-i is, however, biphasic. The fast phase of relaxation is probably an artifact caused by the liquid junction potential of these two solutions (~8 mV), and this will affect the driving force for both leak current and transport current. Thus, Cl-i definition of GAT1 current will have significant artifactual components, particularly with low transport activities, high Cl-i concentrations, and at extreme potentials.
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Figure 4 C shows the typical result obtained for application of 120 mM Na+i in the presence of 20 mM GABAi and 120 mM Cl-i, using NMG+ as the Na+ substitute. In this case, removal of Na+i causes a small inward background Na+ current (<5 pA at 0 mV), which probably reflects Na+-mediated leak current through the patch seal. The activation of GAT1 transport current by Na+i application is somewhat slower than by GABAi and Cl-i. This is because 120 mM Na+i does not "super-saturate" binding sites. The current deactivates faster than with the other substrates because the Na+i dependence of the current is sigmoidal (see Figure 8 B and 9 C). From these results, we conclude that GAT1 transport current is best defined with GABA application and removal, and GABA definition is used in subsequent results.
Failure to detect secondary "gating" or "trans-activation" mechanisms for GAT1.
In experiments similar to those just described, we tested extensively whether the GAT1 transporter undergoes substrate- and time-dependent activity changes via "secondary modulation" or "inactivation" reactions, analogous to reactions identified for cardiac Na+Ca2+ exchange (
Possible existence of Cl-o -independent inward GAT1 current component.
In contrast to the evident tight substrate-current coupling in the activation of reverse (outward) transport current in oocyte patches, forward transport current can be partially activated by extracellular GABA in the nominal absence of extracellular Cl-. Figure 5 shows records from a single patch: (a) the outward current activated by 20 mM GABAi with 120 mM NaCl on both membrane sides. (b) The NO-711i-sensitive inward current with 0.4 mM GABAo, 120 mM Na+o, no Cl-o, and no cytoplasmic substrates. (c) The NO-711i-sensitive inward current with 0.4 mM GABAo, 120 mM NaClo, and no cytoplasmic substrates. The Cl-o-independent inward current (Figure 5b) is more significant at negative membrane potentials (at 120 mV, ~40% of that with 120 mM Cl-o). These results are comparable with results in intact oocytes (
GAT1 reversal potentials.
To further probe substrate coupling by the GAT1 transporter, we measured reversal potentials with various extracellular-to-cytoplasmic substrate ratios and compared them with the expected reversal potentials, Vrev, for 1GABA:2Na+:1Cl- stoichiometry:
where R, T, and F have their usual meanings. For these measurements, IV relations were determined with all substrates on both membrane sides, before and after extracellular application of the high-affinity organic GAT1 inhibitor NO-711 via pipette perfusion. We note that cytoplasmic NO-711 did not block effectively the GAT1-mediated currents under these conditions, presumably because GABA binding sites are occupied on both membrane sides. Figure 6 A shows a typical GAT1 IV relation with a measured Vrev of 45 mV. With the substrate concentrations employed (see Figure 6, legend), the predicted value is 97 mV.
Figure 6 B shows results obtained with a variety of substrate concentrations. The scatter of experimental results is rather large in the repeated measurements, and this reflects the fact that GAT1 current amplitudes in these conditions are rather small. With 120 mM NaCl and 2 mM GABA on both sides, for example, we could not define any GAT1 current. Nevertheless, all of the measured Vrev's, summarized in Figure 6 B, scatter along the theoretical line without systematic deviation. Thus, within our experimental limitations, we find no contradiction to the idea that GAT1 transport is a well-coupled process.
No effect of osmotic gradients on outward GAT1 current.
Since activation of outward GAT1 current with GABA involves changing solution osmolarity by up to 20 mosmol/liter, we tested whether osmotic strength and/or transmembrane osmotic gradients result in any alteration of GAT1 current. We observed no effects of osmolarity changes using dextrose, aspartate, glutamate, or polyethylene glycol (not shown). In a related issue,
Alternating Access Versus Single-file Channel Models of Cotransport
For some ion channels, it is established that ions permeate in a single-file fashion along multiple binding sites (
Figure 7 illustrates predictions for trans- and cis-substrate interactions in the two models (A and D) that we consider fundamental. Predictions for the channel-like model are shown in Figure 7B and Figure C; those for the alternating access model are shown in E and F. We simulated the channel model with our own software using the published parameter values (
We use the simple cotransport model in Figure 7 D to illustrate the equivalent predictions for an alternating access scheme. In this simulation, the cotransporter binds its substrates X and Y instantaneously and independently. The presence of a trans substrate (e.g., Xo) reduces the rate of the "empty carrier translocation" or "return" step (1) in the transport cycle, thereby limiting the rate of transport from the cis side. When the return step is slow, the apparent affinity for cis substrates (e.g., Yi) is higher. The fractional increase of affinity for cis substrates should be proportional to the fractional decrease of maximal transport induced by addition of the trans substrate.
The predictions for X = Na+ and Y = GABA are shown in Figure 7 E, together with the corresponding data from a pipette perfusion experiment. The simulated GABAi dependencies (solid lines) are scaled to the measured magnitudes of outward GAT1 currents with () or without () 100 mM extracellular Na+ at 0 mV. In the presence of high [Na+]o, the maximal GABAi-induced current is decreased, and the apparent affinity of GAT1 for GABAi is increased. The half-maximum GABAi concentrations are 0.90 and 0.52 mM, respectively, in the absence and presence of 100 mM Na+o. Clearly, the data agrees well with the simple alternating access transport model, but not the "channel model" (Figure 7 B).
Figure 7 F shows predictions for cis-substrate interactions by the same alternating access model (Figure 7 D; X = Cl- and Y = GABA), together with the corresponding experimental data. With a rate-limiting empty carrier translocation step in the reverse transport cycle, outward transport current activated by one substrate (e.g., Yi or GABAi) becomes smaller if the concentration of its cis cosubstrate (e.g., Xi or Cl-i) is lowered. This reduction in current amplitude is "overcome" by a sufficiently high concentration of Yi, and a reduction of the cis-cosubstrate concentration results in a decreased apparent affinity for the substrate. This is the case here because the maximal transport rate is determined by the rate of the return step (Figure 7 D, 1), rather than the translocation steps (2) from the cis side. From other observations, we expect that the empty carrier translocation step in GAT1 becomes rate limiting during reverse transport only in the presence of high extracellular Na+. As shown by the data in Figure 7 F with 120 mM extracellular NaCl, reduction of [Cl-]i from 120 to 30 mM shifts the half-maximal concentration for GABAi from 0.3 to 2.7 mM, while the maximal transport current is decreased by only 25%. This pattern is well simulated by the model in Figure 7 D (Figure 7 F, solid lines). In contrast, the channel model predicts that reduction of a cis cosubstrate (i.e., Na+i) increases the apparent affinity for GABAi (Figure 7 C) since GABAi competes with Na+i for entrance to the pore. Thus, both cistrans and transtrans substrate interactions of GAT1 are predicted by alternating access models of cotransport, and they contradict the channel-like model of cotransport. Up to now, we could not envision any modifications of the channel model that would predict the substrate dependencies of the alternating-access model. Also, we point out that our results are consistent with substrates being transported simultaneously, rather than sequentially, as concluded previously by
Effects of Other Extracellular Substrates on Outward GAT1 Current
We have tested whether extracellular GABA at concentrations up to 1 mM affects the outward current in the absence of extracellular Na+, and no clear effect was observed. For extracellular Cl-, we have detected only a small inhibition of outward current (~20%) with 120 mM versus nominally Cl- free extracellular solution in the absence of extracellular Na+.
Outward GAT1 Transport Current: Voltage and Substrate Dependencies
To gain insight into how substrate interactions with GAT1 are affected by membrane potential, we first measured the IV relations of outward GAT1 current over a wide concentration range for one substrate, while leaving the other substrates at fixed high ("saturating") concentrations. A data set for each GAT1 substrate could then be replotted as a series of concentrationcurrent relations at different membrane potentials, and the maximal current amplitude (Imax) and half-maximum concentration (K1/2) for that substrate could be obtained by fitting the concentrationcurrent relations by a Hill equation:
where I is the outward transport current magnitude, [S] is the substrate concentration, and nH is the Hill coefficient.
The corresponding substrate dependencies are shown in Figure 8, whereby the GABAi, Na+i, and Cl-i dependencies of GAT1 outward current at 0 mV are given in AC, respectively. The corresponding voltage dependencies of Imax, K1/2, and nH, are shown in Figure 8DF, respectively. The ImaxV relations are fit by the Boltzmann equation given in MATERIALS AND METHODS (Figure 8, DF, top, solid lines). In each case, the equivalent charge is close to 0.3. Only small changes of IV shapes were detected with changes of substrate concentrations: IV's become slightly less steep with low GABA concentrations and slightly steeper at very low [Na+]i.
The fitted nH and K1/2 are both relatively voltage independent (Figure 8, DF). At membrane potentials between -30 and +90 mV, where measurements are most reliable, nHs are 1.21.4 for Na+i and 0.91.1 for Cl-i. nH for the GABA dependencies was always close to 1, and it was therefore fixed at 1 for this presentation. Over the same range of -30 to +90 mV, the K1/2s are 2.22.8 mM for GABAi, 5383 mM for Na+i, and12.113.6 mM for Cl-i. In the more negative potential range there is a trend for the K1/2's for Na+i and Cl-i to increase.
Outward GAT1 Transport Current: Cytoplasmic Substrate Interactions
Figure 9 describes how GAT1 substrates interact from the cytoplasmic side in the activation of outward transport current. Since the effects of voltage were minor in these interactions, we present only the data at 0 mV. Our protocol was to maintain a high concentration of one of the three substrates, and then systematically examine the effects of lowering one of the other two substrates on the concentration dependence of the third substrate. The transport current in all cases is defined by application and removal of cytoplasmic GABA with otherwise fixed substrate concentrations. In all results, the pipette solution contains 20 mM Cl-, no Na+, and no GABA. The solid lines presented with the data points in Figure 9 represent fits by the functional model we developed for GAT1, which is detailed in another article (
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Figure 9 A shows the cytoplasmic GABA dependencies of transport current. The data points were fit by the Hill equation with nH = 1 to obtain the corresponding Imax's and K1/2's (not shown). When [Na+]i was lowered from 120 to 30 mM with 120 mM cytoplasmic Cl-, the Imax decreased from 155.3 to 30.7 pA, while the K1/2 remained unchanged (2.2 and 2.1 mM, respectively, Figure 9 A, top graph). Similar effects were obtained when reducing cytoplasmic Cl- with 120 mM cytoplasmic Na+ (bottom graph). The Imax is 98.4 pA and the K1/2 is 2.0 mM at 60 mM Cl-i; at 3 mM Cl-i the Imax and K1/2 values are 69.4 pA and 2.9 mM, respectively.
Figure 9 B shows the cytoplasmic Na+ dependencies of the transport current. The top graph shows the effect of changing cytoplasmic GABA from 20 to 2 mM with 120 mM cytoplasmic Cl-: the nH obtained from fit by the Hill equation (not shown) increased from 1.2 to 2.1, the K1/2 for Na+i changed only slightly, and the Imax decreased by 40%. The bottom graph shows the effect of reducing Cl-i from 50 to 5 mM with 12 mM GABA: the K1/2 for Na+i increased by twofold (123 vs. 58 mM), while the nH and the Imax changed only slightly (80 vs. 73 pA).
Figure 9 C shows the cytoplasmic Cl- dependencies of the transport current. These results were fit by the Hill equation with nH = 1 (not shown). The Imax is reduced from 59 pA at 100 mM Na+i to 9.8 pA at 20 mM Na+i, while the K1/2 is increased from 4 to 12 mM. Reduction of [GABA]i also decreases the Imax for Cl- (239 pA at 20 mM GABAi vs. 155 pA at 2 mM GABAi), but the K1/2 increases by only 25% at low [Na+]i (12 mM at 20 mM GABAi vs. 15 mM at 2 mM GABAi).
Only Cl- Can Interact with the Empty GAT1 Transporter from the Cytoplasmic Side
The substrate concentration dependencies presented in the previous section are predicted precisely by a binding scheme for GAT1 cytoplasmic substrates (Scheme 1), where two Na+i and one Cl-i bind sequentially (Cl-iNa+iNa+i) and GABAi binding is independent of Cl-i and Na+i. In this binding scheme, reduction of [Cl-]i will be overcome by high [Na+]i, and GABAi will not affect the apparent affinities of the other cosubstrate. Also, this binding order is mostly consistent with our capacitance data, which indicate that Cl-i interacts with the empty transport in the absence of other cosubstrates (
As described previously, inward transport current can be repeatedly activated in the patch using the pipette perfusion technique (Figure 1). Figure 10A and Figure B, present IV relations of the inward transport current defined by 0.2 mM extracellular GABA in the presence of 120 mM extracellular NaCl. The activation of inward transport current does not require any cytoplasmic ions, nor is inward current enhanced by cytoplasmic ions. As shown in Figure 10 A, the presence of high cytoplasmic Na+ alone (120 mM) has a weak inhibitory effect, and GABA alone (20 mM) has almost no effect. A 2550% inhibition of the inward current is achieved with the combined application of Na+i and GABAi. In contrast, cytoplasmic Cl- strongly inhibits the inward transport current. The inhibition by Cl- is monotonic, and its concentration dependence at 0 mV is shown in Figure 10 C. These data points are from additional measurements in the same patch, and the solid line represents a fit by the equation
where Imax is the current magnitude without Cl-i, and K1/2 is the [Cl-]i at which inward current is reduced by 50%. Inward currents were defined by subtraction of records with and without extracellular GABA. In the presence of 120 mM cytoplasmic Cl-, >90% of the inward current is inhibited. The K1/2 from the fit is 12.4 mM. We also examined the kinetics of inhibition by performing automated concentration jumps, and we resolved no slow components of inhibition that would not be explained by diffusion of Cl-i to and away from the patch (not shown).
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In Figure 11, we have used the capacitance method to further analyze the interactions between GAT1 and its substrates. These results were obtained using 10 kHz/1 mV sinusoidal voltage perturbations. Figure 11 A illustrates the membrane capacitance responses obtained when a patch is rapidly switched from high to 0 Cl- cytoplasmic solution. The magnitude of capacitance increase is larger when the cytoplasmic solution also contains GABA and Na+. Figure 11 B shows the cytoplasmic Cl- dependence of the capacitance change under four conditions: with high GABAi and high Na+i, with high GABAi only, with high Na+i only, and with no GABAi or Na+i. GABA and Na+ alone do not alter much the Cl-i-induced capacitance change. However, the simultaneous presence of cytoplasmic Na+ and GABA results in two changes in the capacitance signal. First, the Cl-i-induced capacitance change is larger at all concentrations. Second, there is a fourfold decrease of the half-maximal Cl-i concentration.
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Together, the results described in Figure 10 and Figure 11 present definitive contradictions to the simple binding scheme for GAT1 cytoplasmic substrates discussed above. First, GABAi alone cannot inhibit the inward current (Figure 10 A) or affect a change of capacitance (Figure 11 B) in the absence of the other substrates. Second, Na+i does not strongly increase the apparent affinity for Cl-i (Figure 11 B, vs.
). An adequate account of this data therefore requires a systematic simulation effort to model GAT1 function, and this is presented in an accompanying article (
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Discussion |
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GAT1 Function Can Be Simple
This study has demonstrated that GAT1-mediated transport currents behave largely as expected for a simple alternating-access cotransporter in the steady state. The Hill slopes of the substrate concentration dependencies and the measured reversal potentials are all consistent with the usual postulated stoichiometry of 1GABA:2Na+:1Cl- for GAT1. Admittedly, the reversal potential measurements show considerable scatter. However, the reverse GAT1 current absolutely requires the presence of GABA and both co-ions. Neither the reverse nor the forward current requires the presence of any trans ions, nor have we observed any trans-stimulation effects of substrates. Our conclusion, that GAT1 can function as a simple transporter with fixed stoichiometry, is similar to that of
Our results are strikingly different from those of
Relations to Previous Oocyte Studies
Two-electrode voltage clamp studies with Xenopus oocytes have previously provided much mechanistic insight into the forward transport mode, which mediates GABA uptake (
Alternating Access versus Channel-like Models of GAT1 Function
The idea that cotransporters might operate in a channel-like fashion, without conformational changes, presupposes that a pore structure could allow hydrophilic molecules as large as GABA to bind specifically and permeate, while disallowing a nonspecific permeation of ions. We know of no clear evidence that this is physically possible, and we have presented two types of results for GAT1 that contradict the channel model (Figure 7). First, addition of extracellular Na+ increases the apparent affinity for cytoplasmic GABA in parallel with a reduction of the maximum reverse GAT1 current. Second, reduction of cytoplasmic Cl- decreases the apparent affinity for GABAi in the presence of extracellular Na+, but not in its absence. In addition, the Cl-i-activated GAT1 capacitance changes become larger and saturate at lower Cl-i concentrations in the presence of both cosubstrates (Figure 11). This indicates that the binding of all three substrates enables further transporter reactions, presumably conformational changes. A final well-known property of alternating access models is that the presence of a substrate on one membrane side can stimulate transport of that substrate from the opposite side. This "self-exchange" of substrates is not predicted by the channel model, but it has been well documented for the GABA transporter (
Complex Cytoplasmic Substrate Interactions
The interactions of cytoplasmic substrates in the activation of the reverse current are not straightforward. As noted above, it is evident that Cl-i can bind in the absence of the other substrates. Furthermore, reduction of Cl-i shifts the Na+i dependence of current to somewhat higher Na+i concentrations, as expected if Na+i binds after Cl-i. However, reduction of [Cl-i] does not shift the dependence of current on GABA, indicating that GABAi probably binds independently from Cl-i. It is then perplexing that GABAi cannot inhibit the inward current. Furthermore, Na+iGABAi interactions are also suggestive of independent binding sites; maximal currents are decreased when either cosubstrate concentration is reduced. That these complexities can be accounted for in the context of a simple transport model (
Physiological Relevance of Cl-i Block
Our observation that cytoplasmic Cl- inhibits potently GAT1 forward transport current raises multiple interesting points. First, since the physiological Cl- concentration inside the oocyte is probably 4050 mM (
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Acknowledgements |
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We express our deep gratitude to Sela Mager for numerous discussions, encouragement, and advice throughout this work. We thank Ernest M. Wright, Lou J. DeFelice, and Baruch I. Kanner for insightful discussions. We thank Ernest M. Wright especially for an open exchange of unpublished data. We thank Sela Mager, Henry Lester, and Eric Schwartz for providing GAT1 constructs. We thank SiYi Feng for expert molecular biological assistance.
This work was supported by a Grant-in-Aid from the American Heart Association.
Submitted: August 10, 1998; Revised: July 1, 1999; Accepted: July 2, 1999.
1used in this paper: GABA, -aminobutyric acid; IV, currentvoltage; MES, 2-(N-morpholino)ethanesulfonic acid; NMG, N-methylglucamine
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References |
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Bechkman, M.L., Quick, M.W. (1998) Probing neurotransmitter transporter function by two-electrode voltage clamp. AxoBits. 22:8-10.
Cammack, J.N., Rakhilin, S.V., Schwartz, E.A. (1994) A GABA transporter operates asymmetrically and with variable stoichiometry. Neuron. 13:949-960[Medline].
Cammack, J.N., Schwartz, E.A. (1996) Channel behavior in a gamma-aminobutyrate transporter. Proc. Natl. Acad. Sci. USA. 93:723-727
DeFelice, L.J., Blakely, R.D. (1996) Pore models for transporters? Biophys. J. 70:579-580[Medline].
Fairman, W.A., Vandenberg, R.J., Arriza, J.L., Kavanaugh, M.P., Amara, S.G. (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature. 375:599-603[Medline].
Galli, A., Blakely, R.D., DeFelice, L.J. (1996) Norepinephrine transporters have channel modes of conduction. Proc. Natl. Acad. Sci. USA. 93:8671-8676
Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M.C., Davidson, N., Lester, H.A., Kanner, B.I. (1990) Cloning and expression of a rat brain GABA transporter. Science. 249:1303-1306[Medline].
He, Z., Tong, Q., Quednau, B.D., Philipson, K.D., Hilgemann, D.W. (1998) Cloning, expression, and characterization of the squid Na+Ca2+ exchanger (NCX-SQ1). J. Gen. Physiol. 111:857-873
Hilgemann, D.W. (1989) Giant excised cardiac sarcolemmal membrane patches: sodium and sodiumcalcium exchange currents. Pflügers Arch. 415:247-249.
Hilgemann, D.W. (1990) Regulation and deregulation of cardiac Na+Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature. 344:242-245[Medline].
Hilgemann, D.W. (1995) The giant membrane patch. In Sakmann B., Neher E., eds. In Single-Channel Recording. New York, NY, Plenum Publishing Corp, 307-327.
Hilgemann, D.W., Lu, C. (1998) giant membrane patches: improvements and applications. Methods Enzymol. 293:267-280[Medline].
Hilgemann, D.W., Lu, C. (1999) GAT1 (GABA:Na+:Cl-) cotransport function: database reconstruction with an alternating access model. J. Gen. Physiol. 114:459-475
Hille, B. (1992) Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 291314.
Jaffe, L.A., Kado, R.T., Muncy, L. (1985) Propagating potassium and chloride conductances during activation and fertilization of the egg of the frog, Rana pipiens. J. Physiol. 368:227-242[Abstract].
Jardetzky, O. (1966) Simple allosteric model for membrane pumps. Nature. 211:969-970[Medline].
Jauch, P., Petersen, O.H., Läuger, P. (1986) Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings. J. Membr. Biol. 94:99-115[Medline].
Jauch, P., Läuger, P. (1986) Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models. J. Membr. Biol. 94:117-127[Medline].
Kanner, B.I., Bendahan, A., Radian, R. (1983) Efflux and exchange of -aminobutyric acid and nipecotic acid catalysed by synaptic plasma membrane vesicles isolated from immature rat brain. Biochim. Biophys. Acta. 731:54-62[Medline].
Kanner, B.I., Schuldiner, S. (1987) Mechanism of transport and storage of neurotransmitters. CRC Crit. Rev. Biochem. 22:1-38[Medline].
Kavanaugh, M.P., Arriza, J.L., North, R.A., Amara, S.G. (1992) Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes. J. Biol. Chem. 267:22007-22009
Keynan, S., Kanner, B.I. (1988) gamma-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes. Biochemistry. 27:12-17[Medline].
Läuger, P. (1991) Electrogenic Ion Pumps. Sunderland, MA, Sinauer Associates, Inc, pp. 2829.
Lester, H.A., Mager, S., Quick, M.W., Corey, J.L. (1994) Permeation properties of neurotransmitter transporters. Annu. Rev. Pharmacol. Toxicol. 34:219-249[Medline].
Loo, D.D., Zeuthen, T., Chandy, G., Wright, E.M. (1996) Cotransport of water by the Na+/glucose cotransporter. Proc. Natl. Acad. Sci. USA. 93:13367-13370
Lu, C.C., Kabakov, A., Markin, V.S., Mager, S., Frazier, G.A., Hilgemann, D.W. (1995) Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp. Proc. Natl. Acad. Sci. USA. 92:11220-11224[Abstract].
Lu, C.C., Hilgemann, D.W. (1999) GAT1 (GABA:Na+:Cl-) Cotransport function: kinetic studies in giant Xenopus oocyte membrane patches. J. Gen. Physiol. 114:445-457
Mager, S., Cao, Y., Lester, H.A. (1998) Measurement of transient currents from neurotransmitter transporters expressed in Xenopus oocytes. Methods Enzymol. 296:551-566[Medline].
Mager, S., Kleinberger-Doron, N., Keshet, G.I., Davidson, N., Kanner, B.I., Lester, H.A. (1996) Ion binding and permeation at the GABA transporter GAT1. J. Neurosci. 16:5405-5414
Mager, S., Naeve, J., Quick, M., Labarca, C., Davidson, N., Lester, H.A. (1993) Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes. Neuron. 10:177-188[Medline].
Matsuoka, S., Hilgemann, D.W. (1994) Inactivation of outward Na+Ca2+ exchange current in guinea-pig ventricular myocytes. J. Physiol. 476:443-458[Abstract].
Meyer, J.S., Shearman, L.P., Collins, L.M. (1996) Monoamine transporters and the neurobehavioral teratology of cocaine. Pharmacol. Biochem. Behav. 55:585-593[Medline].
Misgeld, U., Deisz, R.A., Dodt, H.U., Lux, H.D. (1986) The role of chloride transport in postsynaptic inhibition of hippocampal neurons. Science. 232:1413-1415[Medline].
Ni, Y.G., Miledi, R. (1997) Blockage of 5HT2C serotonin receptors by fluoxetine (Prozac). Proc. Natl. Acad. Sci. USA. 94:2036-2040
Radian, R., Kanner, B.I. (1983) Stoichiometry of sodium- and chloride-coupled gamma-aminobutyric acid transport by synaptic plasma membrane vesicles isolated from rat brain. Biochemistry. 22:1236-1241[Medline].
Risso, S., DeFelice, L.J., Blakely, R.D. (1996) Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells. J. Physiol. 490:691-702[Abstract].
Sonders, M.S., Amara, S.G. (1996) Channels in transporters. Curr. Opin. Neurobiol. 6:294-302[Medline].
Su, A., Mager, S., Mayo, S.L., Lester, H.A. (1996) A multi-substate single-file model for ion-coupled transporters. Biophys. J. 70:762-777[Abstract].
Wong, D.T., Bymaster, F.P., Engleman, E.A. (1995) Prozac (fluoxetine, Lilly 110140), the first selective serotonin uptake inhibitor and an antidepressant drug: twenty years since its first publication. Life Sci. 57:411-441[Medline].
Zühlke, R.D., Zhang, H.-J., Joho, R.H. (1995) Xenopus oocytes: a system for expression cloning and structurefunction studies of ion channels and receptors. Methods Neurosci. 25:67-89.