Lanthanum-mediated modification of GABAA receptor deactivation, desensitization and inhibitory synaptic currents in rat cerebellar neurons

Abstract

1. We investigated La3+ effects on recombinant and native gamma-aminobutyric acid A (GABAA) receptors using rapid agonist applications and on inhibitory synaptic currents (IPSCs) in granule and stellate neurons of rat cerebellar slices. 2. Rapid desensitization of currents elicited by 200 ms pulses of 1 mM GABA to small lifted cells transfected with alpha1beta3gamma2 cDNAs was greatly decreased by the coapplication of 100 microM LaCl3. 3. GABA responses were unaffected when coapplication lasted only 2 ms. In contrast, with LaCl3 pre-perfusion, a significant slowing of deactivation in response to 2 ms applications was observed. LaCl3 pre-perfusion also prolonged the duration of responses to 20 mM taurine. 4. Outside-out patches excised from cells transfected with alpha1beta3gamma2 subunit cDNAs were briefly exposed to a saturating concentration of GABA, eliciting a transient activation of single channel currents with a main conductance of 30 pS. Opening and burst durations increased by pre-equilibration of patches with LaCl3. 5. LaCl3 depressed the peak amplitude without affecting the slow deactivation and desensitization of GABA responses in cells transfected with alpha6beta3gamma2 and alpha6beta3delta cDNAs. No significant difference in La3+ modulation of GABA-gated currents was observed between alpha1beta3gamma2 and alpha1beta3delta receptors. 6. The effects of LaCl3 on deactivation and desensitization of GABA responses observed in nucleated patches excised from rat cerebellar granule and stellate neurons were comparable to those in the cells transfected with alpha1beta3gamma2 cDNAs. In addition, La3+ clearly prolonged the spontaneous IPSC time course without changing the amplitude. 7. Our results indicate that La3+ has a dual action on GABA-gated currents: it decreases desensitization and increases channel opening duration. These actions depend on receptor subunit composition and contribute to the prolongation of IPSCs.

Figure 1. La³⁺ coapplication inhibits rapid desensitization of GABA-gated currents in α1β3γ2-transfected cells

A, averaged response induced by 200 ms application of 1 mM GABA and 1 mM GABA + 100 μm LaCl₃ in a lifted small cell transfected with α1β3γ2 cDNAs. Also shown is the biexponential fitting to the offset currents measured at the end of GABA application with an indication of the fitting parameters. The removal of fast desensitization can be better observed in the right panel where the traces are shown superimposed. B, averaged response in another lifted small cell transfected with α1β3γ2 cDNAs induced by 200 ms application of 1 mM GABA and 1 mM GABA + 100 μm LaCl₃. Applications of GABA + LaCl₃ were preceded by perfusion with LaCl₃ (100 μm). Holding potential, -60 mV. In the right panel the traces are shown superimposed. Above each trace are shown the currents generated by the liquid junction potential due to a 50: 1 dilution of the GABA-containing solution measured after ‘blowing out’ the patch, to give an indication of the duration of the pulse application. Calibration bars apply to A and B, but not to the current pulse. C, summary of the kinetic analysis of the LaCl₃ effects on responses to 200 ms GABA applications. S/P, ratio of the current at the beginning and at the end of the 200 ms application; τ_off,f and τ_off,s, deactivation time constants; %Fast, percentage contribution of the fast component of biexponential functions fitted to the offset currents measured at the end of GABA application. Each bar represents the mean ±s.e.m. of 18 patches studied.

Figure 2. La³⁺ pre-perfusion prolongs GABA current deactivation in α1β3γ2-transfected cells

A, averaged response induced by 2 ms applications of 1 mM GABA and 1 mM GABA + 100 μm LaCl₃ in a lifted small cell transfected with α1β3γ2 cDNAs. Also shown is the biexponential fitting to the current decay with an indication of the fitting parameters. The traces are shown superimposed in the right panel. B, averaged response in another lifted small cell transfected with α1β3γ2 cDNAs induced by 2 ms application of 1 mM GABA and 1 mM GABA + 100 μm LaCl₃. Applications of GABA + LaCl₃ were preceded by perfusion with LaCl₃ (100 μm). Holding potential, -60 mV. Prolongation of the current decay can be better observed in the right panel where the traces are shown superimposed. Above each trace are shown the currents generated by the liquid junction potential due to a 50:1 dilution of the GABA-containing solution measured after ‘blowing out’ the patch, to give an indication of the duration of the pulse application. Calibration bars apply to A and B, but not to the current pulse. C, summary of the kinetic analysis of the LaCl₃ effects on responses to 2 ms GABA applications. τ_f and τ_s, deactivation time constants; %Fast, percentage contribution of the fast component of biexponential functions fitted to the current decay. Each bar represents the mean ±s.e.m. of 18 patches studied.

Figure 3. La³⁺ speed-up recovery from desensitization in α1β3γ2-transfected cells

Left panel in A, superimposed traces evoked by two successive applications in lifted small cells transfected with α1β3γ2 cDNAs of 2 ms GABA pulses separated by 25, 50, 100, 200, 400, 600 and 800 ms intervals. Right panel in A, superimposed traces evoked by two successive applications of 2 ms GABA + LaCl₃ (100 μm) pulses separated by increasing intervals. Holding potential, -60 mV. B, same as in A except that LaCl₃ (100 μm) perfusion preceded the combined GABA + LaCl₃ applications. C and D, comparison of the recovery time course of the second response from the desensitization in the various experimental conditions in a lifted small cell transfected with α1β3γ2 cDNAs. ○, control; •, + La³⁺. In C, La³⁺ was coapplied with GABA, while in D it was also pre-perfused. The percentage recovery from desensitization at each interval between two brief GABA pulses is calculated according to the formula ([Peak2 - onset]/[peak1 - onset]× 100), and is plotted against the interpulse interval. Note the apparent prevention of desensitization with LaCl₃ pre-perfusion. Each data point represents the mean ±s.e.m. of 16 patches studied. The continuous lines are fitted to the double exponential equation: %recovery = 100 - A₁exp(t/τ₁) - A₂exp(t/τ₂) where τ₁, τ₂, A₁ and A₂ are the time constant and the amplitude of the two components. The τ₁, τ₂, A₁ and A₂ values in the respective experimental conditions are indicated. Above each trace are shown currents to give an indication of the duration of the pulse applications. Vertical calibration does not apply to these traces.

Figure 4. La³⁺ prolongs channel opening durations in α1β3γ2-transfected cells

Aa, channel activity evoked by 2 ms pulses of 1 mM GABA as indicated in the current above the traces in an outside-out patch excised from a cell transfected with α1β3γ2 cDNAs. Holding potential, -70 mV. Ab, ensemble averages derived from thirty applications as those shown above. Ac, open time distribution of GABA-activated channel currents for the patch shown above with superimposed double exponential fitting and an indication of the fitting parameters derived. Because of the high level of channel activity as a consequence of the GABA applications we analysed channel currents selecting sweeps with limited channel activity. τ₁ and τ₂ are the decay time constants and A₁ is the percentage contribution of the fast component of biexponential functions fitted to the open time distribution. B, same as A except that GABA was coapplied with LaCl₃ (100 μm) after LaCl₃ pre-perfusion. Summary of the data obtained in 14 patches is given in Table 1.

Figure 5. La³⁺ pre-perfusion prolongs deactivation of taurine-activated currents in α1β3γ2-transfected cells

A, averaged response induced by 2 ms applications of 20 mM taurine and 20 mM taurine + 100 μm LaCl₃ in a lifted small cell transfected with α1β3γ2 cDNAs. Application of GABA + LaCl₃ was preceded by perfusion with LaCl₃ (100 μm). Holding potential, -60 mV. Prolongation of the current decay can be better observed in the right panel where the traces are shown superimposed. B, summary of the LaCl₃ effects on responses to 2 ms taurine applications. τ, deactivation time constants of the monoexponential functions fitted to the current decay. Each bar represents the mean ±s.e.m. of 12 cells studied.

Figure 6. La³⁺ inhibits GABA-gated currents in α6β3δ- and α6β3γ2-transfected cells

A, averaged responses induced in a lifted small cell transfected with α6β3δ cDNAs by 2 ms applications of 1 mM GABA together with the exponential fitting to the current decay and the decay time constant. Fitting was extended to baseline. LaCl₃ (100 μm) coapplied with GABA and pre-perfused, reversibly reduced the peak current. B, averaged responses induced in a lifted small cell transfected with α6β3δ cDNAs by 200 ms applications of 1 mM GABA together with the exponential fitting to the offset currents measured at the end of GABA application and the respective time constant. Fitting was extended to baseline. LaCl₃ (100 μm), coapplied with GABA and pre-perfused, reversibly reduced the peak current. Holding potential, -60 mV. C-E, summary of the LaCl₃ effects on amplitude and kinetics of responses to 2 ms and 200 ms GABA applications in cells transfected with distinct GABA receptor subunit combinations. C, amplitude; D, weighted deactivation time constants (τ_w); E, ratio of the current at the beginning and at the end of a 200 ms application (S/P). Each bar represents the mean ±s.e.m. of 18 patches studied. * Statistical significance with respect to control (P < 0.05 ANOVA followed by t test).

Figure 7. Effects of La³⁺ on GABA responses in nucleated patches from cerebellar granule cells

A, averaged response in a nucleated outside-out patch isolated from a cerebellar granule neuron of a rat at the age of P7, induced by 2 ms applications of 1 mM GABA together with the exponential fitting to the current decay and the weighted time constant. LaCl₃ (100 μm) coapplied with GABA and pre-perfused (middle), produced prolongation of the current decay as it can be better observed in the right panel where the traces are shown superimposed. Left panel in B, superimposed traces evoked by two successive applications of 2 ms GABA pulses separated by 25, 50, 100, 200, 400, 600 and 800 ms intervals in a nucleated patch isolated from a P7 rat cerebellar granule neuron. Right panel in B, superimposed traces evoked in the same patch by two successive applications of 2 ms GABA (1 mM) + LaCl₃ (100 μm) pulses separated by increasing intervals. LaCl₃ was also pre-perfused. C, summary of the effects of LaCl₃ on GABA responses in nucleated patches excised from cerebellar granule cells in rats at P7-P8. Amp, amplitude; τ_f and τ_s, deactivation time constants; %Fast, percentage contribution of the fast component of biexponential functions fitted to the current decay. Each bar represents the mean ±s.e.m. of 18 patches studied. * Statistical significance with respect to control (P < 0.05 ANOVA followed by t test). D, comparison of the recovery time course of the second response from the desensitization in the two experimental conditions in nucleated patches excised from cerebellar granule cells in rats at P7-P8. The percentage recovery from desensitization at each interval between two brief GABA pulses is calculated and plotted as in Fig. 3. The τ₁, τ₂, A₁ and A₂ values in the respective experimental conditions are indicated. Note the apparent prevention of desensitization with LaCl₃ pre-perfusion. Each data point represents the mean ±s.e.m. of 11 patches studied.

Figure 8. La³⁺ prolongs sIPSCs from cerebellar granule cells

A, representative sIPSCs recorded from cerebellar granule neurons at P18. The traces in the left panel are segments of recordings of sIPSCs after 3 min bath perfusion with LaCl₃ (100 μm). B, averages of 100 sIPSCs from the cerebellar granule neurons in A with the biexponential fitting to the current decay and the fitting parameters. C, the cumulative relative frequency of the amplitude and weighted time constants of the sIPSCs shown in panel A is illustrated for control, and LaCl₃ treatment. D, summary of the effects of LaCl₃ on sIPSCs recorded from cerebellar granule neurons at P7-P8 (left) and P18-P20 (middle) and P18-P20 stellate neurons (right). The y-axis labels of panel D are indicated below the x-axis under each pair of histogram columns. Amp, amplitude; τ_f and τ_s, deactivation time constants and percentage contribution of the fast component (%Fast) of biexponential functions fitted to the current decay. Each bar represents the mean ±s.e.m. of 10 cells studied. At least 100 sIPSCs were measured before and during LaCl₃ bath perfusion. * Statistical significance with respect to control (P < 0.05 ANOVA followed by t test).