H+ permeation and pH regulation at a mammalian serotonin transporter

Abstract

The rat serotonin transporter expressed in Xenopus oocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is approximately 10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H+/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H+ by reversal potential measurements. Similar H+-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na+/glucose transporter. The high conductance and high selectivity of the H+-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a "proton-wire mechanism") formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose-response relationships for these two effects suggest that two H+ binding sites, with pKa values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters.

Fig. 1.

Acidic pH induces current in rSERT-injected and uninjected oocytes. A, B, Acidic pH induced current in an rSERT-injected and an uninjected oocyte, respectively. Holding potential, −60 mV. Base solution, Na⁺ Ringer’s solution, pH 7.0. Application of Na⁺ Ringer’s solutions with pH values other than 7.0 is indicated by bars above the current traces.C, Dose–response relationship for the pH-induced current in rSERT-injected oocytes. Current was normalized to the value recorded at pH 3.5 (632 ± 136 nA, mean ± SD,n = 4 oocytes). Vertical barsindicate the SD.

Fig. 2.

Inhibition of the H⁺-induced current.A, The H⁺-induced current in an rSERT-injected oocyte was inhibited by the SERT inhibitors desipramine (10 μm) and fluoxetine (10 μm). Holding potential, −40 mV. Base solution, Na⁺ Ringer’s solution, pH 7.5. B, The H⁺-induced current was partially inhibited in Na⁺ Ringer’s solution compared with the current in NMDG Ringer’s solution. Holding potential, −40 mV.

Fig. 3.

Reversal potential of the H⁺-induced current. A, B, Reversal potential measurements in NMDG Ringer’s solutions at pH 5.5 and 6.5, respectively. The membrane potential was held at −40 mV and jumped for 6 sec to the test potentials noted adjacent to each current trace. Thebars above current traces indicate the period when desipramine (10 μm) was applied. C, Reversal potentials measured by experiments shown above were plotted as a function of pH. Vertical lines indicate the SD (n = 3). Dashed line is the least-square fit, with a slope of −55 mV/pH U.

Fig. 4.

Current–voltage relationship and voltage-jump relaxation kinetics for the H⁺-induced current.A, B, Voltage-clamp recordings from an rSERT-injected oocyte perfused with NMDG Ringer’s solution, pH 5.0, in the presence (A) or absence (B) of 10 μm desipramine. The membrane potential was held at −40 mV and then shifted for 600 msec to a series of test potentials ranging from −140 mV to +40 mV in 20 mV increments. C, Pure H⁺ leakage current obtained by subtracting Afrom B. Dashed line is at zero subtracted current. D, Steady-state currents obtained fromC were plotted as a function of membrane potential. Currents were averaged from the first 50 msec at the test potential.

Fig. 5.

Acidic pH potentiates the 5-HT-induced, transport-associated current. A, 5-HT-induced current recorded in Na⁺ Ringer’s solutions with various pH values. Solution changes are indicated by arrows above the current trace. 5-HT (5 μm) applications are indicated bybars under the current trace. Holding potential, −40 mV. B, 5-HT (5 μm) does not induce the transport-associated current, but rather inhibits the H⁺leakage current in the absence of Na⁺ (NMDG substitution). Holding potential, −40 mV. Base solution, NMDG Ringer’s, pH 7.5.C, Dose–response relationship for the total transport-associated current. The H⁺leakage current and the 5-HT-induced current at each pH value were combined. This combined current was normalized to the maximal current obtained after a nonlinear regression fitting to the Hill equation (dashed line). EC₅₀ = 7.8 ± 1.4 μmH⁺, pH 5.1 ± 0.1, Hill coefficientn = 1.1 ± 0.1 (mean ± SD).Vertical lines indicate the SD (n = 4 oocytes).

Fig. 6.

Effect of pH on [³H]5-HT uptake. Final [³H]5-HT concentration, 1 μm.Vertical lines show SD in measurements from 6 oocytes. Similar results were obtained in at least three separate batches of oocytes.

Fig. 7.

Protons inhibit the transient current.A, Superimposed current traces recorded in Na⁺ Ringer’s solutions with various pH values. Holding potential, −40 mV. During each trial, the oocyte membrane potential was jumped to +60 mV, −140 mV, and +60 mV (protocol is attop). The pH 7.5 solution was tested at both the beginning and the end of the experiment. B, Traces after subtracting the current remaining at pH 5.0 from all other currents recorded at pH >5.0. C, The peak of transient current in B was plotted as a function of [H⁺]. Data were fitted by nonlinear regression to the Hill equation (dashed line). EC₅₀ = 0.49 ± 0.02 μm H⁺, pH 6.31 ± 0.02, Hill coefficientn = 1.06 ± 0.04 (mean ± SD,n = 4 oocytes).

Fig. 8.

Effect of pH on other transporters. Current traces were recorded from oocytes injected with cRNA for GABA (GAT1) (A), Na⁺/glucose (SGLT) (B), dopamine (DAT) (C), or glycine (GLYT1) (D) transporters, or from an uninjected oocyte from the same batch (E). Holding potential, −40 mV. Base solution, NMDG Ringer’s solution, pH 7.5 (left panelsin A–D, and E), or Na⁺Ringer’s solution, pH 7.5 (right panels inA–D). Concentrations of organic substrates wereGABA, 100 μm; glucose (Gluc), 1 mm; dopamine (Dop), 10 μm; glycine (Glyc), 100 μm.