Ionotropic and metabotropic kainate receptor signalling regulates Cl- homeostasis and GABAergic inhibition

Abstract

Key points: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in regulating chloride homeostasis, which is essential for hyperpolarizing inhibition in the mature nervous system. KCC2 interacts with many proteins involved in excitatory neurotransmission, including the GluK2 subunit of the kainate receptor (KAR). We show that activation of KARs hyperpolarizes the reversal potential for GABA (E_GABA ) via both ionotropic and metabotropic signalling mechanisms. KCC2 is required for the metabotropic KAR-mediated regulation of E_GABA , although ionotropic KAR signalling can hyperpolarize E_GABA independent of KCC2 transporter function. The KAR-mediated hyperpolarization of E_GABA is absent in the GluK1/2^-/- mouse and is independent of zinc release from mossy fibre terminals. The ability of KARs to regulate KCC2 function may have implications in diseases with disrupted excitation: inhibition balance, such as epilepsy, neuropathic pain, autism spectrum disorders and Down's syndrome.

Abstract: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl^- ) homeostasis within mature neurons. KCC2 is a secondarily active transporter that extrudes Cl^- from the neuron, which maintains a low intracellular Cl^- concentration [Cl^- ]. This results in a hyperpolarized reversal potential of GABA (E_GABA ), which is required for fast synaptic inhibition in the mature central nervous system. KCC2 also plays a structural role in dendritic spines and at excitatory synapses, and interacts with 'excitatory' proteins, including the GluK2 subunit of kainate receptors (KARs). KARs are glutamate receptors that display both ionotropic and metabotropic signalling. We show that activating KARs in the hippocampus hyperpolarizes E_GABA , thus strengthening inhibition. This hyperpolarization occurs via both ionotropic and metabotropic KAR signalling in the CA3 region, whereas it is absent in the GluK1/2^-/- mouse, and is independent of zinc release from mossy fibre terminals. The metabotropic signalling mechanism is dependent on KCC2, although the ionotropic signalling mechanism produces a hyperpolarization of E_GABA even in the absence of KCC2 transporter function. These results demonstrate a novel functional interaction between a glutamate receptor and KCC2, a transporter critical for maintaining inhibition, suggesting that the KAR:KCC2 complex may play an important role in excitatory:inhibitory balance in the hippocampus. Additionally, the ability of KARs to regulate chloride homeostasis independently of KCC2 suggests that KAR signalling can regulate inhibition via multiple mechanisms. Activation of kainate-type glutamate receptors could serve as an important mechanism for increasing the strength of inhibition during periods of strong glutamatergic activity.

Keywords: Chloride transport; GABA; KCC2; Kainate receptor; electrophysiology; hippocampus; ionotropic; metabotropic.

Figure 1

Kainate receptors modulate IPSCs in CA3 pyramidal cells A, group data showing E _GABA before and after KAR blockade with UBP310 (n = 5). B, group data showing driving force for Cl^– before and after KAR blockade with UBP310 (n = 5). C, plot of voltage–current curve for a cell at t = 0 (E _GABA = −75.1 mV) and t = 5(E _GABA = −79.1 mV). Inset: examples of evoked IPSCs (scale bar = 60 pA, 10 ms). D, group data showing E _GABA at t = 0 and t = 5 (n = 11 control, 8 KA). E, plot of group data showing the effect of 1 μm KA application on E _GABA over time (n = 11 control, 8 KA) F, group data showing resting membrane potential at t = 0 and t = 5 (n = 11 control, 8 KA). G, group data showing driving force for Cl^– at t = 0 and t = 5 (n = 11 control, 8 KA). H, group data showing C^– conductance at t = 0 and t = 5 (n = 11 control, 8 KA). I, group data showing paired pulse ratio at t = 0 and t = 5 (n = 9 control, 8 KA) (^* P < 0.05, ^** P < 0.01).

Figure 2

Kainate receptors activated by 0.1 μm KA modulate IPSCs A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −65.6 mV) and t = 15 (E _GABA = −74.6 mV). Inset: examples of evoked IPSCs (scale bar = 60 pA, 10 ms). B, group data showing E _GABA at t = 0 and t = 15. C, plot of group data showing the effect of 0.1 μm KA on E _GABA over time. D, group data showing resting membrane potential at t = 0 and t = 15. E, group data showing driving force for Cl^– at t = 0 and t = 15. F, group data showing Cl^– conductance at t = 0 and t = 15. G, group data showing paired pulse ratio at t = 0 and t = 15 (n = 9 control, n = 10 KA, all experiments) (^* P < 0.05).

Figure 3

Kainate receptors activated by 1 μM kainic acid modulate IPSCs in the presence of metabotropic signalling blocker NEM A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −59.2 mV) and t = 5 (E _GABA = −73.5 mV). Inset, examples of evoked IPSCs (scale bar: 60 pA, 10 ms). B, group data showing E _GABA at t = 0 and t = 5 (n = 13 control, n = 6 KA). C, plot of group data showing the effect of 1 μm KA on E _GABA over time (n = 13 control, n = 6 KA). D, group data showing resting membrane potential at t = 0 and t = 5 (n = 13 control, n = 6 KA). E, group data showing driving force for Cl⁻ at t = 0 and t = 5 (n = 13 control, n = 6 KA). F, group data showing Cl⁻ conductance at t = 0 and t = 5 (n = 11 control, 8 KA). G, group data showing paired pulse ratio at t = 0 and t = 5 (n = 7 control, 8 KA) (^* P < 0.05) [Correction made on 9 February 2019, after first online publication: Figure 3 was replaced with the current version].

Figure 4

Kainate receptors activated by 1 μM kainic acid modulate IPSCs in the presence of metabotropic signalling blocker GDP‐β‐S A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −68.6 mV) and t = 5 (E _GABA = −74.2 mV). Inset, examples of evoked IPSCs (scale bar: 60 pA, 10 ms). B, group data showing E _GABA at t = 0 and t = 5. C, plot of group data showing the effect of 1 μM KA on E _GABA over time. D, group data showing resting membrane potential at t = 0 and t = 5. with 1 μM KA application E, group data showing driving force for Cl⁻ at t = 0 and t = 5 (n = 13 control, n = 6 KA). F, group data showing Cl⁻ conductance at t = 0 and t = 5 with 1 μM KA application. G, group data showing paired pulse ratio at t = 0 and t = 5 with 1 μM KA application (n = 7 control, n = 6 KA, all 1 μM KA experiments). H, plot of voltage‐current curve for a cell at t = 0 (E _GABA = −67.3 mV) and t = 15 (E _GABA = −69.1 mV) with 0.1 μM KA application. Inset, examples of evoked IPSCs (scale bar: 60 pA, 10 ms). I, group data showing E _GABA at t = 0 and t = 5 with 0.1 μM KA. J, plot of group data showing the effect of 0.1 μM KA on E _GABA over time. (n = 7 control, n = 17 KA, all 0.1 μM KA, experiments) (^* P < 0.05, ^** P < 0.01, ^*** P < 0.001) [Correction made on 9 February 2019, after first online publication: Figure 4 was replaced with the current version].

Figure 5

1 μm or 0.1 μm KA does modulate IPSCs in GluK1/2^–/– CA3 pyramidal cells A, plot of voltage–current curve for a GluK1/2^–/– neuron at t = 0 (E _GABA = −64.4 mV) and t = 5 with 1 μm KA applied (E _GABA = −65.1 mV). Inset: examples of evoked IPSCs (scale bar = 200 pA, 10 ms). B, plot of voltage–current curve for a GluK1/2^–/– neuron at t = 0 (E _GABA = −80.7 mV) and t = 5 with 0.1 μm KA applied (E _GABA = −83.1 mV). Inset: examples of evoked IPSCs (scale bar = 60 pA, 10 ms). C, group data showing E _GABA at t = 0 and t = 5 (n = 6 control, 7 KA 1 μm, 5 KA 0.1 μm).

Figure 6

Kainate receptor mediated modulation of IPSCs activated by 0.1 μm but not 1 μm KA is dependent on KCC2 transporter function A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −68.6 mV) and t = 5 (E _GABA = −71.9 mV). Inset: examples of evoked IPSCs (scale bar = 100 pA, 20 ms). B, group data showing E _GABA at t = 0 and t = 5. C, plot of group data showing the effect of 1 μm KA on E _GABA over time in the presence of specific KCC2 antagonist VU0463271. D, group data showing resting membrane potential at t = 0 and t = 5. E, group data showing driving force for Cl^– at t = 0 and t = 5. F, group data showing Cl^– conductance at t = 0 and t = 5. G, group data showing paired pulse ratio at t = 0 and t = 15. H, plot of voltage‐current curve for a cell at t = 0 (E _GABA = −62.8 mV) and t = 15 (E _GABA = −63.1 mV). Inset, examples of evoked IPSCs (scale bar: 100 pA, 20 ms). I, group data showing E _GABA at t = 0 and t = 15. J, plot of group data showing the effect of 0.1 μM KA on E _GABA over time in the presence VU0463271. (n = 7 control, n = 7 1 μm KA, n = 5 1 μm KA, all experiments) (^* P < 0.05, ^*** P < 0.001).

Figure 7

Kainate receptors activated by 1 μm KA modulate IPSCs in CA3 pyramidal cells in the presence of zinc chelator ZX1 A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −59.3 mV) and t = 15 ( = −65.2 mV). Inset: examples of evoked IPSCs (scale bar = 60 pA, 10 ms). B, group data showing E _GABA at t = 0 and t = 5 (n = 6 control, n = 6 KA). C, plot of group data showing the effect of 1 μm KA on E _GABA over time (n = 6 control, n = 6 KA). D, group data showing resting membrane potential at t = 0 and t = 15 (n = 6 control, n = 6 KA). E, group data showing driving force for Cl^– at t = 0 and t = 5 (n = 6 control, n = 6 KA). F, group data showing Cl^– at t = 0 and t = 15. G, group data showing paired pulse ratio at t = 0 and t = 15 (n = 6 control, n = 5 KA) (^* P < 0.05, ^** P < 0.01).

Figure 8

Kainate receptors activated by 0.1 μm KA modulate IPSCs in CA3 pyramidal cells in the presence of zinc chelator ZX1 A, plot of voltage–current curve for a cell at t = 0 (E _GABA = −59.7 mV) and t = 15 (E _GABA = −63.0 mV). Inset: examples of evoked IPSCs (scale bar = 60 pA, 10 ms). B, group data showing E _GABA at t = 0 and t = 5. C, plot of group data showing the effect of 1 μm KA on E _GABA over time. D, group data showing resting membrane potential at t = 0 and t = 15. E, group data showing driving force for Cl^– at t = 0 and t = 5. F, group data showing Cl^– at t = 0 and t = 15. G, group data showing paired pulse ratio at t = 0 and t = 15 (n = 6 control, 10 KA, experiments) (^* P < 0.05, ^** P < 0.01).