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Originally published In Press as doi:10.1074/jbc.M303735200 on July 28, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38637-38645, October 3, 2003
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Asymmetric Cross-inhibition between GABAA and Glycine Receptors in Rat Spinal Dorsal Horn Neurons*

Yong Li {ddagger} §, Long-Jun Wu {ddagger}, Pascal Legendre ¶ || and Tian-Le Xu {ddagger} || **

From the {ddagger}Department of Neurobiology and Biophysics, University of Science and Technology of China, Hefei 230027 and Institute of Neuroscience, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China and the UMR CNRS 7102 Neurobiologie des Processus Adaptatifs, Université Pierre et Marie Curie, Paris 75252 cedex 05, France

Received for publication, April 9, 2003 , and in revised form, June 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Presynaptic nerve terminals of inhibitory synapses in the dorsal horn of the spinal cord and brain stem can release both GABA and glycine, leading to coactivation of postsynaptic GABAA and glycine receptors. In the present study we have analyzed functional interactions between GABAA and glycine receptors in acutely dissociated neurons from rat sacral dorsal commissural nucleus. Although the application of GABA and glycine activates pharmacologically distinct receptors, the current induced by a simultaneous application of these two transmitters was less than the sum of currents induced by applying two transmitters separately. Sequential application of glycine and GABA revealed that the GABA-evoked current is more affected by glycine than glycineevoked responses by GABA. Activation of glycine receptors decreased the amplitude and accelerated the rate of desensitization of GABA-induced currents. This asymmetric cross-inhibition is reversible, dependent on the agonist concentration applied, but independent of both membrane potential and intracellular calcium concentration or changes in the chloride equilibrium potential. During sequential applications, the asymmetric cross-inhibition was prevented by selective GABAA or glycine receptor antagonists, suggesting that occupation of binding sites did not suffice to induce glycine and GABAA receptors functional interaction, and receptor channel activation is required. Furthermore, inhibition of phosphatase 2B, but not phosphatase 1 or 2A, prevented GABAA receptor inhibition by glycine receptor activation, whereas inhibition of phosphorylation pathways rendered cross-talk irreversible. Taken together, our results demonstrated that there is an asymmetric cross-inhibition between glycine and GABAA receptors and that a selective modulation of the state of phosphorylation of GABAA receptor and/or mediator proteins underlies the asymmetry in the cross-inhibition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although neurotransmission involves specific activation of receptor channels by distinct neurotransmitters, different classes of receptor can be colocalized at the same postsynaptic site and may be activated by the corelease of more than one type of neurotransmitter from the same presynaptic nerve terminal. Recently it has been demonstrated that simultaneous activation of different postsynaptic receptors by the coapplication of their specific neurotransmitter induces cross-modulation of their activation properties. This cross-talk phenomenon has been proposed to represent a fast adaptive process in controlling signal transmission (1). Negative cross-talk was demonstrated between ATP P2X and nicotinic acetylcholine receptors (2–5), between dopamine and adenosine receptors (6), between {gamma}-aminobutyric acid type A (GABAA)1 and dopamine (1) or P2X receptors (7), as well as between dopamine and N-methyl-D-aspartic acid receptors (8). In general, the crosstalk between these receptors is characterized by a partial occlusion of transmitter-evoked currents, i.e. the sum of the amplitudes of responses evoked by each agonist is larger than the amplitude of responses evoked by coapplication of the two neurotransmitters. The molecular mechanism involved in the interaction between P2X and nicotinic acetylcholine receptors is unknown. Negative cross-talk between dopamine and adenosine receptors, between GABAA and dopamine receptors, and between dopamine and N-methyl-D-aspartic acid receptors involves direct intramembranous protein-protein interaction (1, 6, 8), whereas cross-talk between P2X receptor and GABAA receptors (GABAAR) is modulated by chloride efflux and intracellular Ca2+ (7).

In the spinal cord, brain stem (9–12), and cerebellum (13), GABA and glycine can be coreleased by the same synaptic terminal, whereas GABAAR and glycine receptors (GlyR) can be coaggregated at the same postsynaptic site (13, 14). The ionotropic GABAA and GlyR both gate chloride-permeable channels but have distinct structures, resulting from oligomerization of specific subunits (15, 16). There is evidence suggesting that GABAAR and GlyR can interact negatively in mammalian (17, 18) or lamprey (19) spinal cord neurons, in olfactory bulb cells (20), and in the hippocampus (21), i.e. the effects of GABA and glycine are less than additive when they are coapplied. The mechanisms responsible for this cross-inhibition between GABA and glycine remain poorly understood. One possibility is that it reflects the presence, at least in the olfactory bulb, of a receptor subpopulation that can bind either GABA or glycine (20). However the identity of these postulated receptors remains unclear.

In the present study, we explored the mechanisms of the functional interaction between GlyR and GABAAR by analyzing cross-inhibition between glycine- and GABA-induced Cl- currents (IGly and IGABA) in acutely dissociated rat sacral dorsal commissural nucleus neurons, using whole cell, patch clamp recording. Our results showed that cross-inhibition is asymmetric between GABAAR and GlyR and that glycine-induced inhibition of GABA responses depends largely on protein dephosphorylation processes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparation—Rat sacral dorsal commissural nucleus neurons were acutely dissociated according to the method of Wu et al. (11). In brief, pentobarbital-sodium-anesthetized (45–50 mg kg-1, intraperitoneally) Wistar rats (2 weeks old) were decapitated. A segment about 10–15 mm long of lumbosacral (L5-S3) spinal cord was quickly dissected out and immersed in the standard external solution at freezing temperatures. After removing attached dorsal rootlets and the pia matter on the lateral aspects of the cord, the spinal segment was fixed with cyanoacrylic glue to a 15 x 15-mm2 agar block to support the spinal cord tissue. The tissue block was then placed in the cutting chamber of a vibratome tissue slicer (LEICA VT1000S, Leica Instruments Ltd., Wetzlar, Germany). A cold standard external solution (about 4 °C) bubbled with O2 was subsequently placed in the chamber to immerse the tissue block. The spinal segment was sectioned to yield several transverse slices of thickness 400 µm. Slices were preincubated in oxygenated incubation solution for 30 min at room temperature (22–25 °C) and then treated enzymatically in oxygenated incubation solution containing Pronase (1 mg/5 ml) for 20 min at 31 °C. This treatment was followed by exposure to thermolysin (1 mg/5 ml) for 15 min. After the enzyme treatment, slices were kept in enzyme-free incubation solution for 1 h. Then a portion of dorsal horn region was micropunched out and transferred into a culture dish filled with the standard external solution. Neurons were mechanically dissociated with fire-polished Pasteur pipettes under visual guidance under a phase contrast microscope (IX70, Olympus Optical Co., Ltd., Tokyo, Japan). Within 20 min, isolated neurons had attached to the bottom of the culture dish and were ready for electrical recording. The care and use of animals in these experiments followed guidelines and protocols approved by our institutional Animal Care and Use Committee.

Electrophysiology—Whole cell, voltage clamp recordings were made at room temperature (22–25 °C). Culture dishes (Corning 430165, Corning, Inc., Corning, NY) were used as recording chambers and were perfused at 0.5–2.0 ml/min with the standard external solution. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm (Narishige, Tokyo, Japan) on a two-stage puller (PP-830, Narishige), and had a resistance of 4–6 megohms. Membrane potentials were corrected for the liquid junction potential, which had a measured value of 3–4 mV. The series resistance (Rs), estimated from visual cancellation of the capacity transient, was 5–15 megohms. In most experiments, 80–90% series resistance compensation was applied. Artifacts caused by inadequate voltage clamp and space clamp were minimized by selecting for experiment neurons bearing no or short processes. Transmitter-evoked currents recorded from the cell soma normally did not exceed 5 nA. Unless otherwise noted, CsCl solution was used in the recording pipette. After taking these precautions, the largest recorded currents did not differ in time course from the smaller ones. To ensure cell dialysis, data for measurements were obtained at least 3–5 min after the whole cell configuration was established.

Solutions—The composition of incubation solution was (in mM) 124 NaCl, 24 NaHCO3, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose, and it was aerated with 95% O2 and 5% CO2 to a final pH of 7.4. The standard external solution contained (in mM) 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with Tris. The osmolarity of all bath solutions was adjusted to 310–320 mosm/liter with sucrose. The ionic composition of the internal solution medium was (in mM) 120 CsCl, 30 NaCl, 0.5 CaCl22H2O, 1 MgCl26H2O, 5 EGTA, 2 MgATP, and 10 HEPES with the pH adjusted to 7.2. Stocks of MgATP stored at -20 °C were dissolved in the intracellular solution shortly before use to a final concentration of 2 mM. Unless otherwise noted, the membrane potential was held at -50 mV in the voltage clamp studies.

Drugs and Application System—Cyclosporin A (CSPN), staurosporine, and Li4ATP{gamma}S were obtained from Biomol. Okadaic acid (OA) and GF 109203X were from Tocris (Bristol, UK), and H89, KN93, and genistein were from Sigma. All other drugs were from Sigma. Phosphatase inhibitors and protein kinase inhibitors were added to the pipette solution. Agonists or antagonists of GlyR and GABAAR were diluted with extracellular solution to a final concentration and applied via the "Y-tube" method as described previously (22). The tip of the drug tube was positioned between 50 and 100 µm away from the patched neurons. This system allows a complete exchange of external solution surrounding a neuron within 20 ms. Throughout the experiment the bath was perfused continuously with the standard external solution.

Data Acquisition and Analysis—Signals were filtered at 1 kHz, data were collected with an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), which was connected to a Pentium III computer equipped with Digidata 1320A, Clampex, and Clampfit software (Axon Instruments). Data were analyzed with the pCLAMP software (Axon Instruments) and ORIGIN for Windows (Microcal Software, Northampton, MA). Results are presented as the mean ± S.E. (n = number of cells), with statistical significance assessed by Student's t test for two groups' comparison or one-way analysis of variance test for multiple comparisons. A p value of <0.05 or 0.01 was considered statistically significant. To evaluate the strychnine and bicuculline concentrations at half-maximal inhibition of IGly and IGABA (IC50), the mirror image of the Michaelis-Menten equation

(Eq. 1)
was fitted to the data by the least squares method. Current desensitization was fitted by exponential functions, beginning shortly after the peak of response, using Clampfit 8.0 software. Curve fitting was performed using a simplex algorithm least squares exponential fitting routine with single or double exponential equations of the form

(Eq. 2)
where If and Is are the amplitudes of the fast and slow components, respectively; Ip was the amplitude of the nondesensitized current; and {tau}f and {tau}s are respective time constants.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GABA- and Glycine-induced Responses—Cross-talk between GABAAR and GlyR was analyzed on acutely dissociated sacral dorsal commissural nucleus neurons from P14 rats. At this age, neurons express the mature form of GABAAR and GlyR (15, 23). Whole cell recording at the holding potential (Vh) of -50 mV showed that application of GABA or glycine evoked inward chloride currents in all cells tested. The reversal potentials of GABA- and glycine-induced currents (EGABA and EGly) were -2.5 ± 1.1 (S.E., n = 6) and -1.3 ± 0.8 mV (n = 5), respectively, and were not significantly different (p > 0.1; unpaired t test). They were close to the theoretical Cl- equilibrium potential (ECl) calculated from the Nernst equation (-1.3 mV), based on the external and internal Cl- concentration used in the recording (see "Experimental Procedures").

The saturating concentration and EC50 for GABA or glycine were determined by analyzing concentration-response curves for responses to a 10-s application of agonists (Fig. 1). IGly and IGABA were normalized to the peak amplitude of the currents evoked by the application of glycine and GABA at 1 mM, respectively. The normalized data were fitted using a single isotherm function of the form,

(Eq. 3)
where Imax is the maximum current amplitude, I/Imax is the normalized current amplitude, EC50 is the agonist concentration producing 50% of the Imax, and H is the Hill coefficient. This fit produced an EC50 of 8.1 and 34 µM for GABA and glycine, respectively. The Hill coefficients for GABA and glycine were 1.18 and 1.27, respectively.



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  		FIG. 1.
glycine- and GABA-evoked currents in dissociated sacral dorsal commissural nucleus neurons. A1 and B1, sample recordings demonstrating inward currents induced by glycine and GABA at various concentrations. The bars above each trace indicate the drug application period. A2 and B2, concentration-response relationships for IGly and IGABA. IGly and IGABA were normalized to the peak current amplitude induced by 1 mM Gly (A2) and 1 mM GABA (B2), respectively. Each point represents the mean ± S.E. of six to eight neurons. In this and subsequent figures, unless otherwise noted, the Vh was -50 mV.

 

Cross-inhibition between IGABA and IGly—The above experiments suggest that the saturating concentration for GABA or glycine for inducing neuronal responses was >=1 mM. This suggests that responses evoked by the application of 1 mM GABA or 1 mM glycine should be additive if they are induced by the activation of independent receptors. We therefore compared peak amplitudes of currents induced by the application of either GABA or glycine (at 1 mM) and by the simultaneous application of both agonists. As shown in Fig. 2A, coapplication of GABA and glycine evoked currents with an amplitude that was significantly lower than the expected sum (IEX) of currents evoked separately by GABA and glycine (Fig. 2B), suggesting a cross-inhibition between IGABA and IGly (Fig. 2B; n = 36; p < 0.01; t test). A quantitative analysis of this cross-inhibition and its dependence on concentrations of both agonists is shown in Fig. 2C. At 10 µM GABA and glycine, no significant cross-inhibition occurred (91.5 ± 5.3% of the IEX; n = 9; p > 0.05). Significant cross-inhibition was observed at 30 µM GABA and glycine (55.1 ± 6.4% of the IEX; n = 11; p < 0.01), and the effect appears to saturate beyond 30 µM concentrations of the two agonists.



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  		FIG. 2.
Cross-inhibition between IGABA and IGly. A, whole cell currents were induced by application of either 1 mM GABA or 1 mM Gly or by the simultaneous application of 1 mM GABA and 1 mM Gly. All three current traces were obtained from one neuron. The dashed trace represents the expected summation of the IGABA and IGly. B, mean values of 36 experiments shown in A. C, summary of current inhibition data of different concentrations of GABA plus Gly. Ordinates represent IGABA + Gly/(IGABA + IGly) x 100%. In each bar, the concentrations of GABA and Gly were the same. The dashed line indicates (IGABA + IGly)/IEX. In this and subsequent figures, the number of experiments is shown in parentheses. **, p < 0.01.

 

Cross-inhibition Was Not Caused by Nonspecific Receptor Activation by GABA and Glycine—It has been suggested that at high concentrations GABA may activate GlyR (24, 25), resulting in an apparent cross-talk between GABAAR and GlyR (20). We therefore used selective antagonists to determine the specificity of GABA and glycine in their receptor activation (Fig. 3). At 30 µM bicuculline, a selective GABAAR antagonist, the IGABA induced by 1 mM GABA was totally abolished, whereas the IGly induced by 1 mM glycine was unaffected (Fig. 3, A and B). Conversely, 1 µM strychnine, a potent GlyR antagonist, completely abolished IGly without affecting IGABA. Doseresponse curves for bicuculline and strychnine inhibition were obtained from currents evoked by glycine or GABA at 1 mM. The bicuculline inhibited IGABA in a concentration-dependent manner with an IC50 of 0.68 µM (Fig. 3C). Strychnine also inhibited IGABA, but only at concentrations higher than 1 µM (Fig. 3D), with IC50 of 2.83 µM (Fig. 3D). The inhibitory effect of bicuculline on IGly was also evident only at concentrations higher than 30 µM (Fig. 3C), a concentration that completely suppressed IGABA. Strychnine was effective in suppressing IGly at concentrations >= 0.01 µM (Fig. 3D). In contrast, the IC50 values for the inhibition of IGly were 0.04 and 74.2 µM for strychnine and bicuculline, respectively. Taken together, these results suggest that at a concentration of 1 mM, GABA and glycine are unlikely to cross-activate GlyR or GABAAR, respectively, significantly.



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  		FIG. 3.
Effect of bicuculline and strychnine on IGABA and IGly. A and B, 30 µM bicuculline (Bic) was an effective antagonist of 1 mM IGABA but had little effect on 1 mM IGly. To the contrary, 1 µM strychnine (Str) had little effect on 1 mM IGABA but inhibited 1 mM IGly. C and D, concentration-response relationships for inhibition of 1 mM IGABA and 1 mM IGly by bicuculline (C) and strychnine (D), respectively. The antagonists were perfused 30 s before simultaneous application of the agonists and the antagonists. The amplitudes of IGABA and IGly were measured at the peak and expressed as values relative to the control response induced by 1 mM GABA or 1 mM Gly alone, respectively. Each point represents the mean of 6–10 neurons.

 

Cross-inhibition between GABAAR and GlyR Was Asymmetric—A different mode of drug application was used to explore further the interactions between IGly and IGABA and to examine relative contributions of GABAAR and GlyR to the cross-inhibition. As shown in Fig. 4A, GABA or glycine responses obtained in the presence of the other agonist were reduced in amplitude, with GABA-evoked responses more strongly affected by glycine than the reverse condition. The cross-inhibition was also observed during sequential application of 1 mM GABA and 1 mM glycine or vice versa. As shown in Fig. 4B, when glycine was applied before GABA, the amplitude of IGABA became 37.4 ± 2.5% (n = 12; p < 0.01) of the control values observed in the absence of glycine. Similarly, IGly was reduced to 77.9 ± 4.0% (n = 15; p < 0.05) of the control value after a prepulse of 1 mM GABA. Thus the suppression of IGABA by GlyR activation was significantly larger than inhibition of IGly by GABAAR activation (p < 0.01). Fig. 4C summarizes the percent reduction in the mean amplitude of IGABA and IGly when different prepulse concentrations of glycine and GABA were used, respectively. Glycine became effective in cross-inhibition at concentrations >= 30 µM, whereas GABA was effective at 100 µM. This asymmetric cross-inhibition between GABAAR and GlyR did not depend on chloride fluxes or changes in ECl during receptor activation. For ECl close to 0 mV, cross-inhibition was observed at both Vh = -50 and +50 mV (Fig. 5A). Preactivation of GlyR decreased IGABA to 37.4 ± 2.5% of the control at Vh = -50 mV (n = 12) and to 38.5 ± 4.1% of control at Vh = +50 mV (n = 12). Similarly, preactivation of GABAAR decreased IGly to 77.9 ± 4.0% of the control at Vh = -50 mV (n = 10) and to 79.5 ± 4.5% of the control at Vh = +50 mV (n = 10). The differences between percent changes at two Vh values were all not significant (p = 0.1, paired t test). Reversal potentials of response evoked by successive applications of glycine and GABA at 1 mM were estimated by voltage ramp application during the steady-state phase of the evoked currents (Fig. 5B). As shown in Fig. 5C, successive glycine- and GABA-evoked responses had similar reversal potentials. During the glycine prepulse ECl was -4.15 ± 0.4 mV, whereas it was-3.98 ± 0.58 mV during successive GABA application (n = 8).



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  		FIG. 4.
Asymmetric cross-interaction between IGABA and IGly. A and B, sample recordings demonstrating application of 1 mM GABA and 1 mM Gly with two different drug application modes. C, pooled percentage inhibition of IGABA or IGly after Gly (I'GABA in B) or GABA (I'Gly in B) prepulse. Ordinates represent I'GABA/IGABA or I'Gly/IGly x 100%. The dashed line indicates the control IGABA or IGly in the absence of Gly or GABA prepulse. Note the much stronger reduction of IGABA by Gly than contrariwise. In this and subsequent figures, unless otherwise noted, sequential application means that one agonist was applied immediately after (200–400 ms) another agonist application. *, p < 0.05; **, p < 0.01.

 


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  		FIG. 5.
Direction of current flow did not influence cross-inhibition. A, left, inhibition of IGABA by Gly prepulse was similar at -50 mV and at +50 mV (ECl = 0 mV). A, right, the inhibition of IGly by GABA prepulse was similar at -50 mV and at +50 mV. The traces in A were from the same cells. B, representative current trace obtained from the ramp voltage command. The experimental protocol is shown above the current trace. Three voltage ramps ranging from -80 to +80 mV were applied. Gly and GABA were applied to the cell and covered the last two ramps. Traces obtained from the first ramp measured background or leakage currents. C, current-voltage (I-V) curves for IGABA and IGly obtained from traces shown in B.

 

Receptor Channel Activation Is Required for Cross-inhibition—The data shown in Fig. 4 suggest that cross-inhibition depends on the prepulse agonist concentration. However, it remained unclear whether specific receptor channel opening and thus changes in receptor conformation triggered by the prepulse application are required for asymmetric cross-inhibition. To address this issue, we have used specific competitive antagonists for GABAAR and GlyR, bicuculline and strychnine, respectively. Sequential applications of glycine-GABA at 1 mM (Fig. 6A), or GABA-glycine at 1 mM (Fig. 6B) were performed in the presence of 1 µM strychnine and 30 µM bicuculline, respectively, at a concentration that selectively inhibits GlyR or GABAAR completely (Fig. 3, C and D). As shown in Fig. 6, A and B, during sequential applications of transmitters, application of the antagonist known to compete with transmitter binding of the preactivated receptor prevented cross-inhibition. Fig. 6C summarizes the results from all experiments (n = 9) by comparing the amplitudes of control currents (in the absence of prepulses and antagonists) and currents observed after prepulse application, in the absence or presence of either strychnine (for IGABA) or bicuculline (IGly). We found that in the presence of bicuculline, IGly observed after the GABA prepulse had the same amplitude as the control IGly (2.151 ± 0.243 versus 2.213 ± 0.259 with or without bicuculline, p > 0.1). Similarly, in the presence of strychnine, IGABA observed after the glycine prepulse had the same amplitude as the control IGABA (1.615 ± 0.226 versus 1.638 ± 0.204 with or without strychnine, p > 0.1). These data indicate that cross-inhibition between GlyR and GABAAR cannot be explained solely by ligand binding and suggest that activation of receptor channel is required.



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  		FIG. 6.
Functional receptors are required for the current inhibition. A, whole cell currents induced by 1 mM GABA and sequential application of Gly and GABA at 1 mM in the absence or presence of strychnine (Str). B, whole cell currents induced by 1 mM Gly and sequential application of GABA and Gly at 1 mM in the absence or presence of bicuculline (Bic). In the presence of 1 µM strychnine or 30 µM bicuculline, the sequential application of GABA and Gly induced a current with amplitude and kinetics similar to the current induced by GABA or Gly alone. The recordings in A and B were from different neurons. C, statistical data showing the pooled results obtained on nine cells. Strychnine or bicuculline was pretreated for 30 s before simultaneous application with GABA or Gly. NS indicates no statistical significance. *, p < 0.05; **, p < 0.01.

 

Kinetics of Cross-inhibition—Inhibition of IGABA by preactivation of GlyR was accompanied by significant changes in the time course of the desensitizing IGABA component. This was not the case for IGly when GABAAR was preactivated. To examine potential changes in the desensitization of IGly or IGABA during cross-inhibition, time constants of the desensitizing phase of the corresponding current were measured. The desensitization time course of IGly evoked by 10-s applications of 1 mM glycine with or without GABA prepulses could be fitted by the sum of two exponential curves with time constants {tau}f and {tau}s. As shown in Fig. 7, preactivation of GlyR accelerated the desensitization of GABAAR (Fig. 7, A and B). In contrast, preactivation of GABAAR did not change GlyR desensitization kinetics significantly (Fig. 7C).



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  		FIG. 7.
Effect of preapplication of Gly or GABA on IGABA or IGly desensitization. A, 1 mM IGABA-desensitized time course was fitted with the sum of two exponential curves alone. IGABA recorded after 1 mM Gly prepulse displayed a marked decreased in desensitization time constants. B, pooled data obtained on 10 different cells. {tau}f and {tau}s are the fast and slow desensitization time constants of IGABA before (dashed bar) and after Gly prepulse (empty bar). The relative amplitude of {tau}f and {tau}s is shown without (dashed bar) and after the Gly prepulse (empty bar). c/p represents the percentage of nondesensitized IGABA component without (dashed bar) and after Gly prepulse (empty bar) (n = 10). C, pooled data illustrating the desensitization time constants and relative amplitude of IGly without (dashed bar) and after GABA prepulse (empty bar) (n = 26).

 

We have also examined whether cross-inhibition between GlyR and GABAAR was reversible and whether it requires simultaneous activation of the two amino acid receptors. As shown in Fig. 8A, we found that increasing the time interval between sequential applications of glycine and GABA at 1 mM (or vice versa) resulted in a progressively reduced cross-inhibition. The effect of time interval between successive applications of agonist on cross-inhibition was analyzed on 12 and 14 cells for IGABA and IGly inhibition, respectively. Half-inhibition of IGABA was obtained at interpulse intervals close to 41 s (Fig. 8B1). Cross-inhibition cannot be evoked for IGABA for interpulse intervals >=55 s. The time interval for IGly inhibition evoked by GABA prepulses at which no cross-talk occurred was shortened (>=22.5). Half-inhibition of IGly occurred at time intervals close to 14 s (Fig. 8B2).



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  		FIG. 8.
Time-dependent recovery of IGABA or IGly with various intervals between Gly and GABA applications. A1 and A2, illustration of the experimental protocols. Conditioning responses were obtained by applying a saturating concentration of GABA or glycine (1 mM for 10 s). Conditioning pulses were followed at various time intervals from 2 to 60 s by application of Gly or GABA. B1 and B2, normalized amplitude of IGABA, IGly measured at various time intervals after application of Gly (B1, I'GABA, n = 12) or GABA (B2, I'Gly, n = 14).

 

Inhibition of IGABA by GlyR Activation Depends on Phosphatase 2B—It is known that phosphatase 2B (calcineurin) activity can inhibit GABAAR activation (26–28) and modulate GABAAR desensitization kinetics (29, 30). We therefore asked whether phosphatase activity might be involved in the cross-inhibition between GABAAR and GlyR. We first examined the effect of the phosphatase 2B inhibitor CSPN on the asymmetric cross-inhibition elicited by sequential application of 1 mM glycine and GABA at 1-s intervals. When loaded into neurons via the patch electrode, 500 nM CSPN prevented IGABA inhibition by preactivation of GlyR. In contrast, CSPN had no significant effect on the inhibition of IGly evoked by GABAAR activation (Fig. 9, A1 and B). When glycine and GABA were coapplied, 500 nM CSPN blocked cross-talk by 84.6 ± 5.2% (n = 9).



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  		FIG. 9.
Inhibition of phosphatase 2B prevented cross-inhibition. A1, example of currents evoked on the same cell by sequential applications of glycine then GABA (left) or GABA then glycine (right) in the presence of 500 nM CSPN. The same protocol was applied to study the effect of 1 µM OA (A2), 5 µM OA (A3), or 15 mM BAPTA (A4). B, pooled data illustrating percent inhibition of IGABA or IGly after Gly or GABA prepulse in the presence of CSPN, 1 µM or 5 µM OA, or BAPTA. Each bar represents the mean of 10–16 neurons. Note that CSPN and 5 µM OA prevented the inhibition of IGABA by Gly preapplication, whereas 1 µM OA and BAPTA had no effect on the cross-inhibition. **, p < 0.01.

 

Loading the neuron with 1 µM okadaic acid at a concentration that specifically blocks phosphatase 1 and 2A (31) did not prevent IGABA inhibition by preapplication of glycine or IGly inhibition by preapplication of GABA. In contrast, loading the neuron with a concentration of OA (5 µM) known to inhibit phosphatase 2B (30) prevented IGABA inhibition by GlyR preactivation (Fig. 9, A2, A3, and B). OA became nonspecific at concentration higher than 1 µM and mimics the effect of CSPN. Similar to that found for CSPN, 5 µM OA had no significant effect on IGly inhibition evoked by GABA prepulses. Because phosphatase 2B activation may depend on intracellular Ca2+ concentration, we have examined the effect of buffering intracellular Ca2+ with BAPTA. When the neurons were loaded with 15 mM BAPTA via the recording pipette, IGABA was decreased to 39.7 ± 5.3% of its control value, whereas IGly was reduced to 81.2 ± 4.5% of the control following the glycine or GABA prepulse, respectively (Fig. 9, A4 and B). These values did not differ significantly from the cross-inhibition obtained in control conditions (p > 0.1; unpaired t test). Thus the results indicate that changes in intracellular Ca2+ were not directly involved in the cross-inhibition. Inhibition of IGABA, but not of IGly, depends on phosphatase 2B activity, whereas phosphatase 1 and 2A did not appear to be involved.

The level of substrate phosphorylation depends on the action of kinases and phosphatases in the neuronal cytoplasm. Prevention of cross-inhibition by phosphatase 2B inhibition may result from a shift in balance in favor of phosphorylation of the substrate by protein kinases. We first tested this hypothesis by loading the neuron with ATP{gamma}S, which facilitates protein phosphorylation by donating a thiophosphate group in a kinasemediated reaction that resists hydrolysis by phosphatases (32). As shown in Fig. 10, loading the cells with 150 µM ATP{gamma}S (with Li+) prevented IGABA inhibition induced by prepulse activation of GlyR. The effect of ATP{gamma}Son IGABA inhibition increased with time after the onset of whole cell configuration and completely abolished the inhibition within 15 min. The effect is unlikely to result from the loading of Li+ because loading of LiCl (up to 20 mM) did not affect the cross-inhibition (data not shown; n = 4). In contrast to the effect on IGABA cross-inhibition, IGly inhibition evoked by GABA prepulse application was not affected by loading of ATP{gamma}S (Fig. 10).



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  		FIG. 10.
Inhibition of cross-talk by nonhydrolyzable ATP. A, sample recordings demonstrating cross-talk between GABAA and GlyR in the absence (left) and in the presence of intracellular Li4ATP{gamma}S (right). Note that adding ATP{gamma}S in the intracellular recording solution can prevent the inhibition of IGABA by glycine preapplication only. B, pooled data illustrating the percentage of inhibition of IGABA or IGly after glycine or GABA prepulse in the presence of ATP{gamma}S changes with time (n = 6–10). The full effect of ATP{gamma}S on the inhibition of IGABA by glycine prepulse occurred 10 min after the beginning of the intracellular dialysis. Note that ATP{gamma}S had no effect on the inhibition of IGly by GABA preapplication.

 

To confirm further the role of protein phosphorylation, we analyzed the effect of 5 µM staurosporine, a nonselective protein kinase inhibitor, on the cross-inhibition between GABAAR and GlyR (Fig. 11). If changes in GABAAR activation properties resulted from changes in the balance between phosphorylation and dephosphorylation processes, protein kinase inhibition should render cross-inhibition irreversible. The protocol used to determine time interval dependence of cross-inhibition in the presence of staurosporine was identical to that described in Fig. 8. In control conditions, recovery from cross-inhibition occurred in less than 50 s (Fig. 8). However, staurosporine evoked a rundown of IGABA when applied alone. This was not the case for IGly. A 50% rundown of IGABA was found after 40–50 min. To overcome this problem, the effects of staurosporine on cross-inhibition were tested when IGABA amplitude had declined to 50%. Change in GABA response amplitude evoked by glycine preapplication in the presence of staurosporine was compared with change in GABA response amplitude with time in the presence of staurosporine but without preapplication of glycine. As shown in Fig. 11B, IGABA was only slightly decreased 100 s after attaining 50% rundown. Staurosporine cannot block the inhibition of IGABA or IGly evoked by preactivation of GlyR or GABAAR, respectively. However, in these conditions we observed no recovery from IGABA inhibition up to 2 min after evoking cross-inhibition. In contrast, staurosporine had no effect on the recovery of IGly (Fig. 11B). These results indicate that kinase activity is needed for GABAAR but not for GlyR to recover from cross-inhibition. Interestingly, specific antagonists of protein kinase A (H89, 10 µM, n = 6 cells), protein kinase C (GF 109203X, 3 µM, n = 6 cells), tyrosine kinase (genistein, 5 µM, n = 10 cells) or calmodulin-dependent protein kinase II (KN93, 10 µM, n = 10 cells) had no effect on cross-inhibition and its recovery. However, applying a mixture of all four kinase inhibitors mimicked the staurosporine effect on the recovery from cross-inhibition.



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  		FIG. 11.
Inhibition of IGABA by GlyR activation became irreversible after the inhibition of protein kinases. A and B, illustrations of the experimental protocols. A, in the presence of staurosporine (a nonspecific protein kinase), the IGABA decreased with time. To overcome this problem paired application of glycine and GABA was performed when IGABA reached 50% of the control response amplitude (measured at the beginning of the recording). The response amplitudes were then compared with currents recorded on different cells without paired application at the same latency after the beginning on the recordings (empty squares in C). In the presence of staurosporine preapplication of glycine can still inhibit IGABA, but in this case inhibitory cross-talk became irreversible for interpulse intervals up to 2 min. B,in the presence of staurosporine the preapplication of GABA can still inhibit IGABA, but in this case inhibitory cross-talk was still reversible. C, normalized amplitude of IGABA and IGly after glycine or GABA prepulse recorded at various time intervals (n = 6–12). Empty squares are normalized control amplitude values of IGABA without glycine prepulse.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that activation of GABAAR and GlyR in acutely dissociated rat spinal dorsal horn neurons can induce specific asymmetric cross-inhibition of currents induced by GABA and glycine. Our results reveal a novel form of interaction between signal events mediated by two distinct anionic channels and show that specific involvement of intracellular phosphorylation pathways triggered by GlyR activation underlies the asymmetry in the cross-inhibition between the GABAAR and GlyR.

Cross-inhibition Depends on Intracellular GABAAR and GlyR Signaling—Although negative cross-talk between GABAAR and GlyR has been suspected previously (20, 21, 33), the underlying mechanisms remain controversial. Trombley et al. (20) proposed that the interactions between IGABA and IGly in the olfactory bulb reflect direct actions of GABA on GlyR or glycine on GABAAR. This is not the case in rat spinal dorsal horn neurons. In this spinal cord preparation, we found a small minority of neurons lacking either glycine or GABA responses. The currents evoked by GABA and glycine were not affected by prepulse of glycine and GABA, respectively (data not shown). Similar findings were reported for acutely isolated hippocampal neurons (21). In the present study, we have shown that the specific competitive antagonist bicuculline, when applied at a concentration that blocked 100% of IGABA, did not alter IGly. Similarly, IGABA was not modified by strychnine when applied at a concentration that totally suppressed IGly. Furthermore, the inhibition of IGABA by the application of bicuculline prevented the inhibition of IGly by preapplication of GABA. At a prepulse concentration of 1 mM, there is no direct effect of GABA and glycine on GlyR and GABAAR, respectively. Likewise, IGABA was not depressed by prepulse application of glycine in the presence of strychnine. These results indicate that activation of GABAA and glycine receptor channels is required for the cross-inhibition.

What downstream signaling events associated with the receptor channel activation are involved in the cross-inhibition? It is unlikely that alterations in the Cl- gradient resulting from channel activation mediate the cross-inhibition, as proposed previously by Grassi (33). We found that the inhibition remained when IGly and IGABA were changed from inward to outward, indicating that the movement of Cl- is not critical for the inhibition. Furthermore, measurements of agonist-induced Cl- currents with the application of a voltage ramp also showed no change in the reversal potential for the Cl- current (34). These data indicate that cross-inhibition is a receptormediated event unrelated to the Cl- flux across the membrane.

Asymmetry in Cross-inhibition—When GABA and glycine were coapplied, the Cl- current was smaller than the sum of the two individual currents evoked by the application of each agonist. The occlusion level depended on the concentration of the two agonists, and it was nearly maximal when the concentration was >=30 µM. Thus, the intensity of the cross-inhibition between GABAAR and GlyR might be determined by the number of activated receptors, as proposed previously for the interactions between P2X and GABAAR (7).

To determine the respective contribution of the two agonists to the cross-inhibition, we examined the responses evoked by sequential application of these two compounds. Preapplication of glycine more strongly inhibited IGABA than preapplication of GABA did to IGly. The level of current inhibition depended on the concentration of the prepulse agonist, in a manner consistent with that found when GABA and glycine were coapplied. Furthermore, the effect of prepulse glycine on IGABA is accompanied by a decrease in the desensitization time constants of the current response. This was not the case for the effect of GABA prepulse on IGly. Because the effect of desensitization was observed during successive applications of the two agonists, the changes in GABAAR desensitization kinetics by GlyR activation remain even when GlyR is fully deactivated. This effect on the nonactivated GABAAR appears to persist for more than 45 s after the end of the application of glycine. This effect cannot be accounted for simply by cross-desensitization between these two receptors (35) because only GABAAR desensitization properties were modified. These results are also in sharp contrast to the previous reports on the inhibitory crosstalk between ionotropic receptors. No changes in receptor desensitization properties were observed during cross-inhibition between GABAAR and P2X (7) or between P2X and nicotinic acetylcholine receptors (2).

Cross-inhibition of GABAAR by Glycine Depends on Dephosphorylation—Our results indicate that inhibition of IGABA by preapplied glycine involves phosphatase 2B activity, whereas its recovery requires protein kinase activity. Our findings suggest the following model of cross-inhibition: Glycine preapplication results in dephosphorylation by phosphatase 2B and inhibition of IGABA, whereas recovery from inhibition depends on rephosphorylation of GABAAR or of associated proteins. Activation of phosphatase 2B requires the activation of GlyR, but rephosphorylation of GABAAR or of an associated protein does not require the activation of either GlyR or GABAAR. Our observations also indicate that in the absence of glycine prepulse, the balance between basal phosphorylation/dephosphorylation results in stable kinetic properties of the GABAAR during repetitive applications of GABA.

There are three potential mechanisms by which GlyR activation may evoke dephosphorylation by phosphatase 2B. First, activated GlyR may directly activate phosphatase 2B. To our knowledge, there is no existing evidence in support of this possibility. Alternatively, phosphatase 2B is activated indirectly by elevation of the intracellular Ca2+ concentration (27). Although activation of immature GlyR could elevate the intracellular Ca2+ concentration of neurons (36), it is unlikely that glycine application evoked a Ca2+ increase in the present neuronal preparation obtained from mature animal in the voltage clamp mode. Moreover, loading the neuron with BAPTA, a high affinity calcium chelator, did not modify the cross-inhibition between the two receptors. Thus the activation of phosphatase 2B by glycine prepulse does not seem to be mediated by a calcium-dependent process. Finally, GlyR activation may directly change GABAAR conformation through receptor-receptor interaction as proposed previously for some ionotropic receptors as for P2X2 and 5-HT3 receptors (37). This could result in the exposure of phosphorylation sites of GABAAR favorable to phosphatase 2B and/or unfavorable to protein kinase binding.

A large fraction of phosphatase 2B is known to be associated with the plasma membrane (38), a condition favorable for its rapid modulation of membrane receptors. Effectively, phosphatase 2B can modulate GABAAR desensitization kinetics (29) even during outside-out recordings (30). The slow recovery from cross-inhibition may reflect a slower process of rephosphorylation by cytoplasmic protein kinases. However, such a mechanism would require that GlyR is closely associated with GABAAR. Indeed, GABAAR and GlyR are colocalized at inhibitory synapses in the spinal cord and brain stem (10, 39), and it has been demonstrated recently that GABA and glycine can be coreleased from inhibitory synapses in rat sacral dorsal commissural nucleus (11).

Cross-inhibition of GlyR Function by GABA—GABA preapplication did not change GlyR desensitization kinetics, its effect on current amplitude was less pronounced compared with the effect of glycine on IGABA, and the recovery from inhibition was faster for IGly than IGABA. The effects of GlyR subunit phosphorylation on receptor function remain controversial, and they largely depend on subunit combination (15). Our results indicate that inhibition of GlyR activity by GABA is unlikely to result from receptor subunit dephosphorylation or phosphorylation processes because inhibition of phosphatases or protein kinases did not alter the GABA effect on IGly. The most likely mechanism for the inhibition of IGly by GABAAR activation is through direct coupling between these two receptors. However, we cannot exclude the possibility that other unknown cytoplasmic signaling molecules may mediate the functional interaction between these two receptors.

Physiological Significance of the Cross-inhibition—GABA and glycine can be colocalized in the same presynaptic terminals (40) and can be coreleased in several brain regions (9–13), and their associated receptors are colocalized in the postsynaptic density (39), thus providing the physiological conditions for the cross-talk between GABAAR and GlyR. A recent study demonstrating that GABA and glycine interact at the synaptic level is in favor of such a speculation (11). The cross-inhibition of these two major inhibitory neurotransmitter systems could have important functional implications because it may represent an alternative means of limiting excessive inhibition at the synapses where GABA and glycine act as cotransmitters. In particular, GlyR activation and the subsequent phosphatase 2B-dependent GABAAR dephosphorylation would influence the shape and amplitude of GABAergic postsynaptic currents by speeding up GABAAR desensitization kinetics. Indeed, GABAAR desensitization is fast enough to control the time course of GABA-evoked synaptic events (41). Interestingly, a recent work has demonstrated that activity-dependent physical and functional interaction between phosphatase 2B and GABAAR is necessary and sufficient for inducing long term depression at hippocampal CA1 inhibitory synapses (42). Although corelease of GABA and glycine has not yet been detected in the hippocampus, a functional cross-inhibition has been shown for hippocampal GABAAR and GlyR (21). Whether or not the phosphatase 2B-dependent cross-inhibition between GABAAR and GlyR observed in the present study could regulate the synaptic transmission and plasticity remains to be established. Finally, because both GABAAR and GlyR are targets for many clinical therapeutics, it would be of interest to know whether and how drugs acting at one receptor may shift the synaptic influence toward the other transmitter.


   FOOTNOTES
 
* This work was supported by Knowledge Innovation Project Grant KSCX 2-2-04 from the Chinese Academy of Sciences, National Basic Research Program of China Grant G19990 [GenBank] 54000, and National Natural Science Foundation of China Grants 30170247 and 30125015 (to T.-L. X.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ International Brain Research Organization outstanding fellow of 2003. Submitted in partial fulfillment of the requirements for a Ph.D. at the University of Science and Technology of China. Back

|| Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Inst. of Neuroscience, Shanghai Insts. of Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Rd., Shanghai 200031, China. Tel.: 86-21-5492-1751; Fax: 86-21-5492-1735; E-mail: tlxu{at}ion.ac.cn.

1 The abbreviations used are: GABAA, {gamma}-aminobutyric acid type A; ATP{gamma}S, adenosine 5'-O-(3-thiotriphosphate); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CSPN, cyclosporin A; ECl, theoretical Cl- equilibrium potential; EGABA and EGly, GABA- and glycine-induced currents, respectively; GABAAR, GABAA receptor(s); GlyR, glycine receptor(s); IGABA and IGly, GABA- and glycine-induced Cl- currents, respectively; OA, okadaic acid; Vh, holding potential. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Yan-Fang Li and Zeng-You Ye for technique assistance and Drs. Yu-Tian Wang and Mu-ming Poo for helpful comments on this manuscript.



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 DISCUSSION
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