News Physiol Sci 19: 80-84, 2004;
doi:10.1152/nips.01504.2003
1548-9213/04 $5.00
News in Physiological Sciences, Vol. 19, No. 2, 80-84,
April 2004
© 2004 Int. Union Physiol. Sci./Am. Physiol. Soc.
Electrophysiological Insights into the Mechanism of Ion-Coupled Cotransporters
Antonio Peres,
Stefano Giovannardi,
Elena Bossi and
Riccardo Fesce
Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, and Center for Neurosciences, University of Insubria, 21100 Varese, Italy
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Abstract
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Most ion-coupled cotransporters display, in the absence of organic substrate, transient currents resembling the gating currents of voltage-dependent ion channels. Detailed comparison of these currents in different ionic and temperature conditions with the corresponding steady-state currents when translocation of the substrate occurs reveals new insights into the mechanisms of the process.
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Introduction
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Many small-sized hydrophilic organic molecules of biological relevance, such as nutrient amino acids, osmolites, and neurotransmitters, cross the cell membrane via specific proteins, powered by the electrochemical gradient of accompanying co-ions. For a long time, the electrophysiological study of this process has been hampered by the very low amplitude of the currents generated by this activity, which did not allow this field to thrive in a way comparable with the strictly related one of ion channels. In the past 12 years, however, a significant number of ion-coupled cotransporters for several kinds of substrates (neurotransmitters, amino acids, sugars, peptides, and nucleotides) have been cloned (12, 19, 20). Overexpression of these cDNAs in heterologous systems, especially Xenopus laevis oocytes, has allowed precise electrophysiological measurements, which have revealed new and unexpected features in the behavior of transporters.
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Most cotransporters share similar electrophysiological properties
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In addition to the current associated with the translocation of the organic molecule (a definite expectation for electrogenic transporters), another, unanticipated, and particularly interesting type of current is observed when the organic substrate is not present: the so-called "pre-steady-state current," an intramembrane charge movement seen as a transient current in response to voltage or Na+ concentration jumps (15, 17). This kind of current is observed to varying degrees in the majority of the cloned transporters, quite independently of the gene family to which they belong and also irrespective of the chemical nature of the organic substrate transported (4, 13, 17, 18).
Different models have been proposed to account for the new electrophysiological observations emerging from the experiments on heterologously expressed transporters. Two rather extreme possibilities are depicted in Fig. 1
: as proposed by Loo et al. (16) for the glucose transporter SGLT1, the charges moving in the membrane field, and giving rise to the pre-steady-state currents, belong to the protein (Fig. 1A), whereas Lester et al. (15) proposed for the GABA transporter rGAT1 that the charge movement is due to the displacement of ions between the extracellular space and a cavity in the transporter (Fig. 1B). Another hypothesis may be conceived in which both intrinsic and extrinsic charges may be involved in the process, and some models do not even require a conformational change to explain charge movement, nor to support active transport, as exemplified in those based on multisubstrate, single-file electrodiffusion (6, 21).
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FIGURE 1. Two possible models that may account for the different features exhibited by cotransporters. A: ordered kinetic model proposed for the human glucose transporter SGLT1 (redrawn from Ref. 16). In this case, the pre-steady-state currents are produced by rearrangement of the empty transporter between states 1 and 6, involving the displacement of an intrinsic negative charge (blue circles). Binding of positive ions (red circles) neutralizes the transporter, transport-associated current is generated by the 6-to-1 passage. The green hexagon represents glucose. B: alternating-access model proposed for the GABA transporter rGAT1 (redrawn from Ref. 15). In this case, the intramembrane charge movement is due to the in-and-out movement of ions (red circles) and/or to a strictly associated conformational change; binding of GABA (blue rectangles) leads to prompt release of both substrates and return to state 1.
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Pre-steady-state currents and gating currents
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An example of pre-steady-state and transport-associated currents generated by voltage jumps in the neuronal GABA transporter rGAT1 is shown in Fig. 2, A and B. Pre-steady-state currents share several characteristics with the gating currents of voltage-dependent ionic channels: 1) they are transient; 2) the area under the transient at the "on" of the voltage pulse is equal to that of the "off"; and 3) they show saturation at extreme positive and negative voltages. These are all landmarks of intramembrane charge movement, as it has been shown to occur in the voltage sensor for the opening of the ionic channels (2). However, there are also dissimilarities that it is important to point out. The first difference concerns size: whereas the gating currents of ion channels are orders of magnitude smaller than the transmembrane current that flows through the open channel, in the case of transporters, the pre-steady-state and the transport-associated currents are on the same order of magnitude (see Fig. 2, A and B). The gating currents of channels are best measured in the absence of the normally permeating ions, and therefore the charges sustaining gating and transmembrane currents are clearly distinct. In cotransporters, on the contrary, the ions that are required to activate the transport of substrate are required for the generation of the pre-steady-state currents as well; indeed, the two kinds of current appear to be mutually exclusive; that is, whereas the pre-steady-state current can be best observed in absence of substrate, it disappears when the substrate is present in saturating amounts (4, 17, 18).
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FIGURE 2. Pre-steady-state (A) and transport-associated currents (B) in response to voltage steps from a -40-mV holding potential to membrane voltages ranging from -120 mV to +40 mV in an oocyte expressing the neuronal GABA transporter rGAT1. C: amount of charge in the inner transporter position (Qin), from integration of the transients in A. D: transport-associated current (Itr, squares, from traces in B). The product Qin•r (circles) is very close to the experimentally determined Itr (squares). E: relaxation rate (r) of the transients in A (at 22°C). F: negative shift induced by higher temperature on the relaxation rate. Adapted from Refs. 3 and 7.
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The properties of the gating currents of ion channels are clearly consistent with a voltage-dependent conformational change of the channel protein, displacing intrinsic charges in the membrane field (2): a small movement of gating charges might give rise to a much larger current through the open pore; furthermore, the gating current may occur even when the permeating ions are not present. The pre-steady-state current of cotransporter may also be explained as due to rearrangement of a charged portion of the protein in the membrane field (16, 17); however, their peculiar characteristics outlined above may suggest a second possibility: that is, the charge relocation might be due to the motion of ions that move back and forth between the external solution and an open vestibule in the transporter facing the extracellular side (15, 17).
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Pre-steady-state currents and transport-related currents
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In any case, there is little doubt that pre-steady-state currents represent a partial step in the transport cycle, and therefore a quantitative relation is expected between the parameters of charge movement and the current associated with the transport of the organic substrate. Clearly all models, such as those illustrated in Fig. 1
, lend themselves to simulations that may be more or less satisfactory. Often, however, the relatively high number of states does not allow an analytical solution, preventing the derivation of simple relations between pre-steady-state and transport currents and making it more difficult to describe the process in an intuitive way. We have recently discovered (7, 10) that in the neuronal GABA transporter rGAT1 the transient currents in the absence of GABA may be simply related to the stationary currents generated during translocation of the neurotransmitter. In this transporter we have seen that at any voltage the following relation holds:
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where Itr is the amplitude of the transport-associated current in the presence of saturating GABA, Qin is the amount of charge displaced at an inner transporter position, and r represents the relaxation rate of the pre-steady-state current. The coincidence between experimental and calculated curves in Fig. 2D demonstrates the validity of Eq. 1, which has been further tested in our lab in a variety of other conditions (7, 10).
This experimentally derived relation leads immediately to the quite intuitive physical counterpart shown in Fig. 3: indeed, Eq. 1 describes the behavior of a simple electrical circuit made of a resistor in series with a capacitor, the capacitor possibly being short-circuited by the presence of GABA. The simple derivation in Fig. 3 shows that the product of Q and r, the charging parameters of the capacitor when the switch is open, corresponds to the current flowing through the resistor when the switch is closed. It appears therefore that the charges displaced to an inner position of the transporter in the absence of GABA (giving rise to the pre-steady-state currents) are converted to an equivalent amount of transmembrane current-carrying charge when GABA is present, i.e., they switch from a capacitive to a conductive behavior. It may be further noted that the electrical circuit of Fig. 3 is remarkably similar to the cartoon shown in Fig. 1B, proposed by Lester et al. (15) for the same transporter, and therefore this scheme is strongly favored.
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FIGURE 3. A: electrical analog of rGAT1. The switch "GABA" is open in absence of GABA; therefore voltage jumps will displace a charge Q = CV on the capacitor plates, with rate r = 1/ = 1/RC. Hence the product Q•r is equal to V/R, i.e., to the current flowing in the circuit when the switch is closed by the presence of GABA. B: time course of the relevant electrical quantities in response to a step voltage change across the membrane.
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These results, together with a more detailed analysis of the behavior of the pre-steady-state and transport-associated current in nonsaturating GABA conditions (7), may be described by the simplified three-state kinetic scheme illustrated in Fig. 4A. The model is simple enough to be treated analytically, giving a set of equations that have been successfully compared with the experimental results (7).
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FIGURE 4. A: 3-state kinetic scheme sufficient to simulate the experimental observations and to justify relation 1. In the absence of GABA the transporter may exist in states T0 and T1, the latter being the state with charge (circles) in an inner transporter position.  and ß are the voltage-dependent unidirectional rate constants for the passage from state T1 to state T0 and vice versa, respectively. The model requires that GABA (G; rectangle) may bind only when the transporter is already loaded with the ions, i.e., to state T1, with binding constant k1. The model further assumes that when the fully loaded state of the transporter is reached (T2), immediate release of all substrates to the cytosol and prompt return to the empty state T0 will occur. This scheme leads to a 3-differential-equations system that can be easily solved to produce the relations indicated in the figure. B: pictorial representation of the transporter states; red circles represent ions, and green rectangle represents GABA; see text for details.
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Pre-steady-state currents and structure
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The cartoons of Fig. 1 suggest that the transporter might present a sort of vestibule or cavity open to the extracellular solution and accessible to ions and organic substrate. Detailed knowledge of the three-dimensional structure of cotransporters still awaits the preparation of suitable crystals and X-ray diffraction analysis. To date, the only information available comes from bacterial transporters and concerns bacteriorhodopsin, the bacterial oxalate transporter OxlT (resolved by high-resolution cryomicroscopy; Refs. 14 and 22), and the lactose permease of Escherichia coli (described at a resolution of 3.5 Å by X-ray diffraction; Ref. 1). In all three cases, a disposition of transmembrane helices suggesting the presence of an ample hydrophilic cavity has been observed. Furthermore, structure-activity analysis based on cysteine-scanning mutagenesis on the glutamate transporter GLT-1 (11) has led the authors to propose a membrane topology, including a complex arrangement of transmembrane segments, conformationally sensitive reentrant pore loops, and linker regions in the carboxy-terminal half of the protein. This intricate structure is certainly compatible with the idea of a relatively wide opening facing the extracellular solution.
Indeed, this representation is supported by several electrophysiological observations on the pre-steady-state currents. Our laboratory has shown (8) that mutation K448E in rGAT1, a residue located in the fifth extracellular loop (according to 12-transmembrane-segment topology models), induces voltage-dependent shifts and pH sensitivity in the pre-steady-state currents, effects that may be interpreted as due to changes in the apparent Na+ concentrations in a restricted volume close to or inside the transporter structure. Further interesting observations concern the effects of Cl-: lowering the external concentration of this ion produces a shift of the pre-steady-state currents toward more negative membrane potentials (5); oddly enough, although opposite in charge, Na+ causes qualitatively similar effects. Evidently the combined Boltzmann-Hill approach used by Mager et al. (17) to explain the Na+ effects is not sufficient to account for the Cl- action, and a mechanism by which Na+ and Cl- interfere with one another, each favoring the other’s ability to bind to the transporter, must be conceived. One possibility, which we have put forward (5), is that a Donnan-like system might exist between bulk external solution and a vestibule in the transporter, just preceding the charge-translocation machinery.
Another suggestion in this direction comes from studies on the effects of temperature on these currents. In two reports concerning SGLT1 (13) and rGAT1 (3), it was observed that, besides accelerating the relaxation of the transients, increasing temperatures produced a negative shift in the charge vs. voltage and on the rate vs. voltage relationships (see Fig. 2F). Analysis in terms of unidirectional rate constants revealed that temperature has a stronger effect on the outward rate compared with the inward rate (3), and calculation of the corresponding activation energies suggested that although the outward movement of charges probably requires a conformational change, the inward movement is diffusion limited. One might envisage therefore a diffusional entry of ions into a vestibule, followed by the inward charge movement and by a conformational change of the transporter locking the ions in position; conversely, the outward motion of ions would first require a conformational unlocking and therefore would be more sensitive to temperature (Fig. 4B).
All of these considerations seem to point then to the existence of a relatively ample, but possibly somehow selective, opening in the transporter portion facing the extracellular side. This prediction, based on physiological observations, will need to be validated, or refuted, by structural analysis.
As stated at the beginning, pre-steady-state and transport-associated currents are quite general features of electrogenic cotransporters; we have shown that for the neuronal cotransporter rGAT1 a straightforward relation links these two currents, suggesting a simple and intuitive physiological mechanism. In other transporters, even from the same genic superfamily, the situation does not appear to be so clear cut: for example, in the human norepinephrine transporter the pre-steady-state currents persist in the presence of the organic substrate (9), suggesting that they are due to the movement of an intrinsic charge. The case of rGAT1 probably represents a fortunate situation in which a combination of characteristics has allowed simplification of an otherwise more complicated kinetic scheme to only three essential states. Indeed, the neutral nature of the organic substrate (which does not contribute to the transport current), the absence of counterflux of other ions, and the functional voltage range and slow kinetics are all features that allow easier and more precise determinations of the parameters in rGAT1 compared with other transporters.
This transporter may then represent a particularly simple case possessing the basic mechanism, over which further specializations may be developed by other transporters to account for more particular properties.
A pictorial exemplification of the kinetic scheme of Fig. 4A is tentatively illustrated in Fig. 4B: in state T0, only ions (red circles) can interact with the transporter; following diffusional entry into the vestibule and charge displacement, a conformational change takes place that 1) locks the ions in the inner position, preventing their intracellular dispersion, 2) makes GABA access possible, and 3) must be reversed to let the charges go back to the extracellular solution, justifying the stronger temperature dependence of the outward charge movement (see above). This is then state T1, from which the situation may move to T2 if a GABA molecule happens to interact with the transporter before a spontaneous return to T0 takes place. To satisfactorily simulate the experimental results, the kinetic scheme of Fig. 4A requires state T2 to have a very brief lifetime; therefore, the substrates promptly dissociate toward the cytosol and the transporter returns immediately back to state T0.
An interesting additional consequence of the model in Fig. 4A is a purely kinetic definition of the apparent GABA affinity. In fact, it can be shown (7) that the GABA concentration giving rise to half of the maximal current at each potential (K1/2) is given by
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in which k1 is the binding constant of GABA to state T1; that is, the GABA apparent affinity depends on the rate of charge movement, which in turn depends on Na+ concentration and voltage. It is worthwhile to note that in a mammalian presynaptic membrane at about -60 mV and 37°C, considering the negative shift produced by higher temperatures (Fig. 2F and Ref. 3), the rate r will be close to its minimum, and therefore the apparent affinity for GABA will be at its maximum, optimizing the removal of neurotransmitter from the synaptic cleft.
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Acknowledgments
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Work in our laboratory was funded by grants from the Ministry of Education, University, and Research (Projects of National Interest), from University of Insubria Project of Excellence Program, and from Fondazione Cariplo.
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Copyright © 2004 by the Int. Union Physiol. Sci./Am. Physiol. Soc.
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Introduction
Most cotransporters share...
