Analysis of macroscopic ionic currents mediated by GABArho1 receptors during lanthanide modulation predicts novel states controlling channel gating

Abstract

Lanthanide-induced modulation of GABA(C) receptors expressed in Xenopus oocytes was studied. We obtained two-electrode voltage-clamp recordings of ionic currents mediated by recombinant homomeric GABArho(1) receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents.GABA-evoked chloride currents were potentiated by La(3+), Lu(3+) and Gd(3+) in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration.Lu(3+) also induced receptor desensitization and decreased the deactivation rate of GABArho(1) currents. In the presence of 300 microM Lu(3+), dose-response curves for GABA-evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABArho(1) receptors was modulated by Lu(3+). These results suggest that the potentiation of the anionic current was the result of a direct lanthanide-receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABArho(1) channels gating are proposed.

Figure 1

Potentiation of GABA-induced Cl⁻ currents by lanthanides. (a) Representative trace of an ionic current elicited by 3 μM GABA and potentiated by 1 mM La³⁺. La³⁺ was applied during the maximum response to GABA. The effect was sustained during the presence of lanthanide and fully reversible. (b and c) Same experiment as shown in (a) but 1 mM Lu³⁺ (b) or 1 mM Gd³⁺ (c) was applied instead of La³⁺. The time course for lanthanide action in (b and c) differed from that observed in (a). The fast initial increase in current amplitude was followed by a rapid decrease of the effect in the presence of lanthanide (and GABA). After removing the lanthanide (b and c), a rebound was observed.

Figure 2

Lu³⁺-induced GABA-activated Cl⁻ currents potentiation depends on the type and concentration of agonist used. Representative currents evoked by 0.3 and 1 μM GABA or 1 mM β-alanine, recorded in the same oocyte, were superimposed to compare time courses after a 1 mM Lu³⁺ application, and their amplitudes were normalized. Desensitization of GABAρ₁ responses was more prominent for the higher GABA concentration and for currents evoked by β-alanine.

Figure 3

Analysis of Lu³⁺ effects on GABAρ₁ receptors. (a) Dose–response curves for GABA in the presence or absence of 300 μM Lu³⁺. Response amplitudes were expressed as fraction of 30 μM GABA-evoked currents. (b) Potentiation of GABA-induced Cl⁻ currents exerted by different concentrations of Lu³⁺. Data were obtained by normalizing the current amplitude to control responses (1 μM GABA). Inset: Superimposed current traces showing the effects of increasing concentrations of Lu³⁺ on currents evoked by 1 μM GABA in a representative experiment. (c) GABA concentration dependence of the Lu³⁺ potentiation of GABA-induced Cl⁻ currents. (d) I–V relationship for GABAρ₁ responses evoked by 1 μM GABA in the presence or absence of 300 μM Lu³⁺.

Figure 4

Lanthanide-induced slowing of GABA-induced Cl⁻ currents deactivation. Representative recordings illustrating the effect of 1 mM Lu³⁺ on currents evoked by 1 μM GABA. Dotted current trace: control experiment, Lu³⁺ and GABA were removed (dotted bars represents applications time course) once the current reached a peak. Solid current trace: lanthanide was present during GABA removal from the bath (solid bars represents applications time course). Current traces were superimposed for simplicity. Inset: Deactivation constants measured for control currents or during a constant application of 1 mM Lu³⁺ during GABA washout.

Figure 5

Modulation by Lu³⁺ of GABAρ₁ receptor antagonism. (a) Representative experiments showing the effect of Lu³⁺ on the time course and degree of antagonism exerted by TPMPA on GABA-induced Cl⁻ currents. In these experiments, 300 μM Lu³⁺ was added alone (dotted trace) or together with 30 μM TPMPA (solid trace) during the plateau of GABA-induced Cl⁻ currents evoked by 1 μM GABA. (b) Same experiment as shown in (a) but TPMPA antagonism was tested on responses evoked by 3 μM GABA (dotted trace), a concentration that elicited a response equivalent to that obtained by 300 μM Lu³⁺. (c and d) Same experiments as shown in (a and b) but 3 μM picrotoxin was applied instead of TPMPA. Recovery of GABA-induced Cl⁻ currents after antagonism was omitted in this protocol.

Figure 6

Model of GABAρ₁ receptor gating during lanthanide modulation. (a) Kinetic schemes representing a mechanism for homomeric GABAρ₁ receptor gating during lanthanide action, where A₃R^*L corresponds to a second open state and D represents a desensitized state. Constants describing transitions in the proposed model are listed in the Table 1. (b) Peak open probability (curves) as a function of GABA concentration or GABA plus 300 μM Lu³⁺. Note that these simulations parallel experimental results (points; control: filled squares, 300 μM Lu³⁺ filled circles, the same as illustrated in Figure 3a). (c) Numeric simulations of the experiment shown in Figure 4. Dotted trace: application of 1 mM Lu³⁺ during 1 μM GABA and deactivation rate after removal of GABA and Lu³⁺. Solid trace: the model reproduced the slower deactivation time course observed experimentally during a constant application of 1 mM Lu³⁺ during GABA washout.