Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters

Abstract

The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.

Fig. 1.

Annotated amino acid sequences for rSERT and related transporters. A, Alignment between rSERT and hSERT (GenBank accession numbers X63253 and L05568, respectively). Putative membrane topology is indicated by the blue(intracellular), white (transmembrane, N terminal near the intracellular face), pink (extracellular), andgray (transmembrane, N terminal near the extracellular face) regions. Residues in rSERT are shown in dark blue letters; residues in hSERT (shown below rSERT inblack) are dashed where identical to those in rSERT and shown explicitly where the two sequences differ, but for simplicity, differences are not shown for the N- and C-terminal intracellular regions (22 and 4 differences, respectively). Positions of conserved restriction enzyme sites that were used for chimera construction are marked by the enzyme name. Residues in which mutations have been studied in the present experiments are marked by ↓.Numbers mark the positions in which mutations were characterized (Figure legend continues)in previous studies: 1, C109 (Chen et al., 1997a); 2, I172, Y176, and I179 (Chen et al., 1997b); 3, N177 (Lin et al., 1996); and 4, S545 (Sur et al., 1997). The C109A mutant was used for some of the experiments in this paper (see Figs. 6, 7). TM, Transmembrane. B, Summary of the membrane topology shown in A. C, Alignments for 19 transporters, including ∼44 residues in the putative extracellular region studied in this paper, flanking the E493 region of rSERT. Initially, a total of 65 Na⁺, Cl⁻-dependent neurotransmitter transporters and orphan transporters was selected from the GenBank database. A candidate sequence (the human sequence if possible) was then selected from each cluster and was realigned. The rSERT residues mutated in this study are shown by ↓.

Fig. 2.

Effects of acidic pH on transport-associated current in wild-type (WT) rSERT and hSERT. Note that pH 5.5 solution produces an additional inward current in the absence of 5-HT in both transporters (Cao et al., 1997). However, only in rSERT the pH 5.5 solution also increases the transport-associated current during 5-HT application. The holding potential was −40 mV. The base solution was normal Na⁺ Ringer’s solution, pH 7.4. Applications of acidic Na⁺ Ringer’s solution, pH 5.5, and 5-HT (3 μm) are indicated by horizontal bars.

Fig. 3.

Voltage dependence of the transport-associated current at pH 5.5. A, Data for a representative oocyte expressing rSERT. Row 1, Voltage-clamptraces in the absence of 5-HT, pH 5.5. The membrane potential was held at −40 mV and jumped to test potentials at 10 mV increments between +40 mV and −100 mV for 100 msec. Row 2, Traces for the same voltage-clamp protocol applied 3 min later in the presence of 5-HT (3 μm), pH 5.5. Row 3, Subtraction revealing the 5-HT-induced current at pH 5.5. Note that the subtracted traces are displayed at a higher gain. B, Data for a representative oocyte expressing hSERT, with voltage-clamp protocols and subtraction described in A. The oscillations in the final epoch ofrow 3 are an artifact of the algorithm that suppressed 60 Hz interference earlier in the traces.C, The transport-associated current at pH 5.5 plotted versus membrane potential. The current was averaged over the final 30 msec of the test pulse. Data are mean ± SEM for five rSERT and four hSERT oocytes.

Fig. 4.

Localization of a region responsible for pH dependence of the transport-associated current. Experiments similar to those of Figure 2 were performed on chimeric rSERT–hSERT transporters. The constructs used restriction sites forSacI, BglII, and BamHI. For locations of each restriction enzyme site, see Figure 1. Note that all the data are consistent with the interpretation that pH 5.5 enhances the transport-associated currents in transporters containing C-terminal sequences from rSERT. The BamHI construct contains the fewest rSERT residues (155) and indicates that the sequences governing pH 5.5 enhancement are located in the C-terminal 155 residues.

Fig. 5.

Effects of acidic pH on transport-associated current from the point mutants rS483F (A), rT490K (B), rH572Y (C), hK490T (D), E493Q (E), and E494Q (F) and the double point mutant E493Q–E494Q (G). Recording conditions are described in the legend to Figure 2.

Fig. 6.

Comparisons between transport-associated current in C109A and C109A–E493C.A, B, Effects of acidic pH on transport-associated current in typical traces from C109A (A) and C109A–E493C (B). Recording conditions are described in the legend to Figure 2. C, D, Effects of cysteine-modifying reagents MTSEA and MTSES on transport-associated current from C109A (C) and C109A–E493C (D). Transport-associated currents were recorded both before and after MTS-reagent treatments and were normalized to the current recorded before treatment. Error bars represent the SD in measurements from three oocytes.

Fig. 7.

Functional effects of the E493C mutation.A, Comparison of C109A and C109A–E493C on the ratio of net charge movement to net 5-HT uptake. Error bars represent the SD in measurements from three oocytes. B, Comparison of C109A–E493C and a control (C109A) on the Na⁺dependence of 5-HT uptake. NMDG was used as a substitute for Na⁺. Error bars represent the SD in measurements from six oocytes. C, Dose–response relationship for the transport-associated current. Data were fit by nonlinear regression to the Hill equation. D, Voltage-dependent transient current in the C109A–E493C mutant and inhibition of this current by 5-HT. Current traces recorded in the presence (+) and absence (−) of 5-HT (3 μm) are superimposed. The voltage protocol is shown at the top.

Fig. 8.

Characterization of the E493D mutant.A, Effect of acidic pH on transport-associated current. Recording conditions are described in the legend to Figure 2.B, Transient current in E493D and inhibition of this current by 5-HT. Current traces recorded in the presence and absence of 5-HT (3 μm) are superimposed. The voltage protocol is the same as that shown in Figure 7D.C, Comparison of E493D and WT on the ratio of net charge movement to net 5-HT uptake. Error bars represent the SD in measurements from three oocytes.

Fig. 9.

Schematic diagram of the transport cycle for SERT. Six numbered states are enclosed within an oval;arrowheads on the oval denote the direction of normal progression through the transport cycle. Fully coupled stoichiometry applies within the oval; one 5-HT molecule is transported into the cell along with a single Na⁺ ion and a single Cl⁻ ion (states 6, 1, 2, and 3). A single K⁺ ion leaves (states 5 and 6). The external gate is labeled in red when open; the internal gate is labeled in blue when open. When 5-HT, Na⁺, and Cl⁻ bind within the lumen, the external gate closes, and the internal gate opens (state 1 → state 2). This changes the compartmentalization of the three substrates, which accounts for their coupled flux. When K⁺ then binds within the lumen, the internal gate closes, and the external gate opens, accounting for the obligatory role of K⁺(state 4 → state 5). Outside the oval is the state (2*) corresponding to the uncoupled transport-associated current. We hypothesize that this uncoupled transport-associated current represents a violation of the alternating-access rules; both gates are open simultaneously. Retaining the convention from the ion channel literature, we describe this conducting state by an *. The red arrows indicate probabilities, not formal rate constants; E493Q, E493C, and low pH increase the probability of state 2*, whereas E493D decreases this probability.