Neuronal Chloride Regulation via KCC2 Is Modulated through a GABA_BReceptor Protein Complex

Abstract

GABAB receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABAB receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABAB receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABAA receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABAB receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel “crosstalk” between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABAA receptors (GABAARs) and metabotropic GABAB receptors (GABABRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABABR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABAAR. We demonstrate that GABABR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABAAR. Our data therefore support an additional mechanism by which GABABRs are able to modulate fast synaptic inhibition.

The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

“…The 800 kDa complex also contained a prominent subnetwork of proteins associated with synaptic signaling and organization (GABBR1, GABBR2, GRM2, and GRM3). Importantly, the GABAB receptor has previously been demonstrated to interact with KCC2 directly (35), which further substantiates our findings. For approximately half of the proteins in both the 600 and 800 kDa complexes, there were no known interactors other than KCC2.…”

“…This could be either due the transient or low affinity nature of these interactions, or that these interactors are less abundant in complex with KCC2 than the interactors presented here, as unbiased methodologies provide information on associated proteins in rank order of abundance. LC-MS/MS further confirmed the veracity of our purification strategy as these high molecular weight species contained GABABRs, which has been previously shown to be associated with KCC2 (35). Interestingly we identified gephyrin associated with KCC2 in the 800 kDa band along with CYFIP1/2, NCKAP1 and CNTN1 and other proteins that are enriched at inhibitory synapses (51,52).…”

KCC2 regulates neuronal excitability and hippocampal activity via interaction with Task-3 channels

“…We then investigated the mechanisms underlying Task-3 down-regulation upon KCC2 silencing. KCC2 has been shown to interact with a variety of proteins, including transmembrane proteins such as GluK2 44 , Neto2 45 and GABA B receptors 46 that regulate KCC2 membrane expression or the intracellular guanylyl exchange factor β PIX which is regulated through direct interaction with KCC2 35,36 . We therefore asked whether KCC2 might also interact with Task-3 channels and thereby influence their membrane expression and/or function.…”

“…KCC2 interacts with a variety of transmembrane as well as intracellular partners, including postsynaptic receptors 44,45,46 , actin-related proteins 34,35,36 and others involved in protein trafficking and recycling 37 . Although these interactions are most often considered with regard to KCC2 expression or function, they also influence the function of KCC2 partners.…”

Chloride dynamics alter the input-output properties of neurons

“…Rat organotypic hippocampal slice cultures were prepared using a method similar to that described (40,41) and that KCC2 is the major active Cltransporter in these neurons as EGABA is affected by KCC2-blocking drugs, but not by NKCC1-blocking drugs (42).…”