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Intracellular chloride concentration influences the GABAA receptor subunit composition.

Succol F, Fiumelli H, Benfenati F, Cancedda L, Barberis A - Nat Commun (2012)

Bottom Line: The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons.We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively.Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova 16163, Italy.

ABSTRACT
GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

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Zolpidem and THIP reveal changes of α1-δ subunit expression in altered KCC2 expression.(a) Summary of the average percent increase in the sIPSC decay kinetics in KCC2 and KCC2 shRNA transfected neurons by application of Zolpidem (at DIV6–7 control n=6; KCC2 over n=9 *P<0.05; ANOVA; at DIV12–13 control n=9; KCC2 shRNA n=10; **P<0.01; ANOVA). (b) Representative whole-cell responses in KCC2 and KCC2 shRNA transfected neurons to applications of THIP (30 μM) and GABA (1 mM). (c) Summary of the average current density ITHIP/IGABA in KCC2 transfected neurons (control, grey bar n=22; KCC2 over, black bar n=22; ***P<0.001 ANOVA). (d) Summary of the average current density ITHIP/IGABA in KCC2 shRNA transfected neurons (control, grey bar n=12; KCC2 shRNA, white bar n=12; ***P<0.001 ANOVA). Data is presented as mean±s.e.m.
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f6: Zolpidem and THIP reveal changes of α1-δ subunit expression in altered KCC2 expression.(a) Summary of the average percent increase in the sIPSC decay kinetics in KCC2 and KCC2 shRNA transfected neurons by application of Zolpidem (at DIV6–7 control n=6; KCC2 over n=9 *P<0.05; ANOVA; at DIV12–13 control n=9; KCC2 shRNA n=10; **P<0.01; ANOVA). (b) Representative whole-cell responses in KCC2 and KCC2 shRNA transfected neurons to applications of THIP (30 μM) and GABA (1 mM). (c) Summary of the average current density ITHIP/IGABA in KCC2 transfected neurons (control, grey bar n=22; KCC2 over, black bar n=22; ***P<0.001 ANOVA). (d) Summary of the average current density ITHIP/IGABA in KCC2 shRNA transfected neurons (control, grey bar n=12; KCC2 shRNA, white bar n=12; ***P<0.001 ANOVA). Data is presented as mean±s.e.m.

Mentions: To further demonstrate that modulation of KCC2 protein levels affects the expression GABAAR subunits, we used a pharmacological approach exploiting Zolpidem, an allosteric modulator of GABAARs that when applied at low concentration preferentially enhances current mediated by α1-containing receptors1819, and THIP, a GABAAR agonist, which selectively activates δ-containing GABAARs20. As Zolpidem has been reported to selectively prolong the decay and increase the peak amplitude of synaptic α1-containing GABAA receptors1819, its potentiating effect on synaptic currents reflects the relative abundance of the α1 subunit in synaptic GABAARs. The effect of Zolpidem was studied only on the sIPSC decay kinetics, since it has been demonstrated that the amplitude enhancement exerted by this drug critically depends on the extent of postsynaptic receptor saturation18. Spontaneous GABAergic activity was recorded in the presence of Zolpidem (300 nM) in both DIV6–7 and DIV12–13 cultures under conditions of altered KCC2 expression. At DIV6–7, the increase of sIPSC mean decay time constant induced by Zolpidem application was significantly higher in KCC2-transfected cells with respect to control (5.0±3.5 and 18.7±6.8 ms in control and KCC2, respectively; P<0.05 ANOVA) (Fig. 6a). In contrast, the robust Zolpidem-induced relaxation of sIPSC decay kinetics was significantly reduced in KCC2-shRNA transfected neurons (44.6±5.9 and 17.9±4.4 ms in control and KCC2 shRNA, respectively; P<0.01 ANOVA) (Fig. 6a). These data suggest that the expression levels of KCC2 affect α1 subunit expression. To directly test whether up- or down-regulation of KCC2 also interferes with δ subunit expression we exogenously applied both GABA and THIP pulses and, as previously reported, the ratio between the current peak amplitude obtained in THIP and GABA (ITHIP/IGABA) was taken as a measure of the expression of δ-containing GABAARs920. At DIV6–7, KCC2 over-expression significantly increased the ITHIP/IGABA ratio (0.05±0.02 and 0.16±0.05 in control and KCC2 over, respectively; P<0.001 ANOVA), indicating an increase in δ-containing GABAARs with respect to control (Fig. 6b,c). Conversely, KCC2 shRNA reduced the ITHIP/IGABA ratio (0.26±0.05 and 0.05±0.01 in control and KCC2 shRNA, respectively; P<0.001 ANOVA) indicating that KCC2 down-regulation significantly decreased the expression of extrasynaptic tonic receptors at mature stages (Fig. 6b,d). Taken together, these results suggest that KCC2 expression affects GABAAR subunit composition at the level of both α1 (phasic current) and δ (tonic current) subunits.


Intracellular chloride concentration influences the GABAA receptor subunit composition.

Succol F, Fiumelli H, Benfenati F, Cancedda L, Barberis A - Nat Commun (2012)

Zolpidem and THIP reveal changes of α1-δ subunit expression in altered KCC2 expression.(a) Summary of the average percent increase in the sIPSC decay kinetics in KCC2 and KCC2 shRNA transfected neurons by application of Zolpidem (at DIV6–7 control n=6; KCC2 over n=9 *P<0.05; ANOVA; at DIV12–13 control n=9; KCC2 shRNA n=10; **P<0.01; ANOVA). (b) Representative whole-cell responses in KCC2 and KCC2 shRNA transfected neurons to applications of THIP (30 μM) and GABA (1 mM). (c) Summary of the average current density ITHIP/IGABA in KCC2 transfected neurons (control, grey bar n=22; KCC2 over, black bar n=22; ***P<0.001 ANOVA). (d) Summary of the average current density ITHIP/IGABA in KCC2 shRNA transfected neurons (control, grey bar n=12; KCC2 shRNA, white bar n=12; ***P<0.001 ANOVA). Data is presented as mean±s.e.m.
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f6: Zolpidem and THIP reveal changes of α1-δ subunit expression in altered KCC2 expression.(a) Summary of the average percent increase in the sIPSC decay kinetics in KCC2 and KCC2 shRNA transfected neurons by application of Zolpidem (at DIV6–7 control n=6; KCC2 over n=9 *P<0.05; ANOVA; at DIV12–13 control n=9; KCC2 shRNA n=10; **P<0.01; ANOVA). (b) Representative whole-cell responses in KCC2 and KCC2 shRNA transfected neurons to applications of THIP (30 μM) and GABA (1 mM). (c) Summary of the average current density ITHIP/IGABA in KCC2 transfected neurons (control, grey bar n=22; KCC2 over, black bar n=22; ***P<0.001 ANOVA). (d) Summary of the average current density ITHIP/IGABA in KCC2 shRNA transfected neurons (control, grey bar n=12; KCC2 shRNA, white bar n=12; ***P<0.001 ANOVA). Data is presented as mean±s.e.m.
Mentions: To further demonstrate that modulation of KCC2 protein levels affects the expression GABAAR subunits, we used a pharmacological approach exploiting Zolpidem, an allosteric modulator of GABAARs that when applied at low concentration preferentially enhances current mediated by α1-containing receptors1819, and THIP, a GABAAR agonist, which selectively activates δ-containing GABAARs20. As Zolpidem has been reported to selectively prolong the decay and increase the peak amplitude of synaptic α1-containing GABAA receptors1819, its potentiating effect on synaptic currents reflects the relative abundance of the α1 subunit in synaptic GABAARs. The effect of Zolpidem was studied only on the sIPSC decay kinetics, since it has been demonstrated that the amplitude enhancement exerted by this drug critically depends on the extent of postsynaptic receptor saturation18. Spontaneous GABAergic activity was recorded in the presence of Zolpidem (300 nM) in both DIV6–7 and DIV12–13 cultures under conditions of altered KCC2 expression. At DIV6–7, the increase of sIPSC mean decay time constant induced by Zolpidem application was significantly higher in KCC2-transfected cells with respect to control (5.0±3.5 and 18.7±6.8 ms in control and KCC2, respectively; P<0.05 ANOVA) (Fig. 6a). In contrast, the robust Zolpidem-induced relaxation of sIPSC decay kinetics was significantly reduced in KCC2-shRNA transfected neurons (44.6±5.9 and 17.9±4.4 ms in control and KCC2 shRNA, respectively; P<0.01 ANOVA) (Fig. 6a). These data suggest that the expression levels of KCC2 affect α1 subunit expression. To directly test whether up- or down-regulation of KCC2 also interferes with δ subunit expression we exogenously applied both GABA and THIP pulses and, as previously reported, the ratio between the current peak amplitude obtained in THIP and GABA (ITHIP/IGABA) was taken as a measure of the expression of δ-containing GABAARs920. At DIV6–7, KCC2 over-expression significantly increased the ITHIP/IGABA ratio (0.05±0.02 and 0.16±0.05 in control and KCC2 over, respectively; P<0.001 ANOVA), indicating an increase in δ-containing GABAARs with respect to control (Fig. 6b,c). Conversely, KCC2 shRNA reduced the ITHIP/IGABA ratio (0.26±0.05 and 0.05±0.01 in control and KCC2 shRNA, respectively; P<0.001 ANOVA) indicating that KCC2 down-regulation significantly decreased the expression of extrasynaptic tonic receptors at mature stages (Fig. 6b,d). Taken together, these results suggest that KCC2 expression affects GABAAR subunit composition at the level of both α1 (phasic current) and δ (tonic current) subunits.

Bottom Line: The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons.We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively.Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova 16163, Italy.

ABSTRACT
GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

Show MeSH
Related in: MedlinePlus