Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons.

KCC2 is a neuron-specific K(+)-Cl(-) cotransporter that is essential for Cl(-) homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl(-) extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.

Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain.

KCC2 is the neuron-specific member of the of K(+)-Cl(-) cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl(-) from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl(-), which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K(+)-Cl(-) cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl(-) through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl(-) regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K(+)-Cl(-) cotransport in neurons.

Extracellular potassium regulates the chloride reversal potential in cultured hippocampal neurons.

Inhibitory GABAergic synaptic transmission in the mammalian hippocampus depends upon a hyperpolarized reversal potential for Cl(-) (ECl). To examine the regulation of ECl hyperpolarization we cultured hippocampal neurons for two weeks in either a low- or a high-concentration of KCl (2.6 or 18.7 mM, respectively). Neurons were then recorded from standard extracellular solution containing 3 mM K+, using the dual perforated patch clamp technique. Low-KCl cultured neurons fired spontaneous action potentials (APs; 0.33+/-0.11 Hz), while high-KCl cultured neurons were quiescent, resulting in a significant difference in AP activity (p=0.042). This high-KCl-induced decrease in activity was accompanied by depolarizations of both the AP threshold (p<0.001) and ECl (p<0.001), and a decrease in input resistance (IR, p<0.001), when compared with low-KCl cultured neurons. Blocking AP firing of low-KCl neurons during culturing with 1 muM tetrodotoxin did not alter ECl hyperpolarization, when compared with drug-free cultured low-KCl neurons (p=0.627); thus AP firing is not required for ECl hyperpolarization. Acute perfusion of a high-KCl extracellular solution onto low- or high-KCl cultured neurons demonstrated that high-KCl significantly depolarized the resting membrane potential (RMP). The KCl-induced change in ECl did not correspond with alterations in the expression of the cation chloride cotransporters KCC2 and NKCC1, as determined by western blotting (p=0.736). These findings suggest that: (1) extracellular K+ regulates ECl hyperpolarization; and, (2) the use of high-KCl during neuronal culture produces biophysically abnormal parameters, and thus should be discouraged.

Kainate receptors coexist in a functional complex with KCC2 and regulate chloride homeostasis in hippocampal neurons.

KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

GABAergic synaptic transmission regulates calcium influx during spike-timing dependent plasticity.

Coincident pre- and postsynaptic activity of hippocampal neurons alters the strength of gamma-aminobutyric acid (GABA(A))-mediated inhibition through a Ca(2+)-dependent regulation of cation-chloride cotransporters. This long-term synaptic modulation is termed GABAergic spike-timing dependent plasticity (STDP). In the present study, we examined whether the properties of the GABAergic synapses themselves modulate the required postsynaptic Ca(2+) influx during GABAergic STDP induction. To do this we first identified GABAergic synapses between cultured hippocampal neurons based on their relatively long decay time constants and their reversal potentials which lay close to the resting membrane potential. GABAergic STDP was then induced by coincidentally (±1 ms) firing the pre- and postsynaptic neurons at 5 Hz for 30 s, while postsynaptic Ca(2+) was imaged with the Ca(2+)-sensitive fluorescent dye Fluo4-AM. In all cases, the induction of GABAergic STDP increased postsynaptic Ca(2+) above resting levels. We further found that the magnitude of this increase correlated with the amplitude and polarity of the GABAergic postsynaptic current (GPSC); hyperpolarizing GPSCs reduced the Ca(2+) influx in comparison to both depolarizing GPSCs, and postsynaptic neurons spiked alone. This relationship was influenced by both the driving force for Cl(-) and GABA(A) conductance (which had positive correlations with the Ca(2+) influx). The spike-timing order during STDP induction did not influence the correlation between GPSC amplitude and Ca(2+) influx, which is likely accounted for by the symmetrical GABAergic STDP window.