Tarek Z. Deeb

NMDA receptor activity downregulates KCC2 resulting in depolarizing GABAA receptor mediated currents

KCC2 is a neuron-specific K+-Cl− co-transporter that maintains a low intracellular Cl− concentration that is essential for hyperpolarizing inhibition mediated by GABAA receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca2+ influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

Modulation of neuronal activity by phosphorylation of the K–Cl cotransporter KCC2

The K-Cl cotransporter KCC2 establishes the low intraneuronal Cl- levels required for the hyperpolarizing inhibitory postsynaptic potentials mediated by ionotropic γ-aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs). Decreased KCC2-mediated Cl- extrusion and impaired hyperpolarizing GABAAR- and/or GlyR-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity. Recent evidence suggests that the intrinsic ion transport rate, cell surface stability, and plasmalemmal trafficking of KCC2 are rapidly and reversibly modulated by the (de)phosphorylation of critical serine, threonine, and tyrosine residues in the C terminus of this protein. Alterations in KCC2 phosphorylation have been associated with impaired KCC2 function in several neurological diseases. Targeting KCC2 phosphorylation directly or indirectly via upstream regulatory kinases might be a novel strategy to modulate GABA- and/or glycinergic signaling for therapeutic benefit.

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

Tonically active GABAA receptors in hippocampal pyramidal neurons exhibit constitutive GABA-independent gating

Phasic and tonic inhibitory currents of hippocampal pyramidal neurons exhibit distinct pharmacological properties. Picrotoxin and bicuculline methiodide inhibited both components, consistent with a role for GABAA receptors; however, gabazine, at a concentration that abolished miniature GABAergic inhibitory postsynaptic currents and responses to exogenous GABA, had no effect on tonic currents. Because all GABA-activated GABAA receptors in pyramidal neurons are gabazine-sensitive, it follows that tonic currents are not GABA-activated. Furthermore, picrotoxin-sensitive spontaneous single-channel events recorded from outside-out patches had the same chord conductance as GABA-activated channels and were gabazine-resistant. Therefore, we hypothesize that GABAA receptors, constitutively active in the absence of GABA, mediate tonic current; the failure of gabazine to block tonic current reflects a lack of negative intrinsic efficacy of the antagonist. We compared the negative efficacies of bicuculline and gabazine using the general anesthetic propofol to directly activate GABAA receptors native to pyramidal neurons or α1β3γ2 receptors recombinantly expressed in human embryonic kidney 293 cells. Propofol activated gabazine-resistant, bicuculline-sensitive currents when applied to either preparation. Although gabazine had negligible efficacy as an inhibitor of propofol-activated currents, it prevented inhibition by bicuculline, which acts as an inverse agonist inhibiting GABA-independent gating. Recombinant α1β1/3γ2 receptors also mediated agonist-independent tonic currents that were resistant to gabazine and inhibited by bicuculline. Thus, gabazine is a competitive antagonist with negligible negative efficacy and is therefore unable to inhibit GABAA receptors that are active in the absence of GABA because of either anesthetic or spontaneous gating. Moreover, spontaneously active GABAA receptors mediate gabazine-resistant tonic currents in pyramidal neurons.

Common determinants of single channel conductance within the large cytoplasmic loop of 5-HT3 and α4β2 nicotinic acetylcholine receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4′), 436 (0′), and 440 (4′) residues by 5-HT3B (-4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β2 nAChRs (γ = 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

The KCC2 cotransporter establishes the low neuronal Cl− levels required for GABAA and glycine (Gly) receptor‐mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non‐synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C‐terminus, exhibit significantly impaired Cl−‐extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EGly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.

Structural Determinants of Ca2+ Permeability and Conduction in the Human 5-Hydroxytryptamine Type 3A Receptor

Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased PCa/PCs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca2+, the single channel conductance of the 5-HT3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca2+. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca2+ on macroscopic currents mediated by 5-HT3A receptors. We introduced a D293A mutation into the 5-HT3A(QDA) receptor (yielding the 5-HT3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca2+. The D293A mutation decreased the PCa/PCs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca2+. The D293A mutation also reduced PCa/PCs when engineered into the wild-type 5-HT3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence PCa/PCs and additionally directly demonstrated a depression of single channel conductance by Ca2+.

Possible alterations in GABAA receptor signaling that underlie benzodiazepine‐resistant seizures

Benzodiazepines have been used for decades as first‐line treatment for status epilepticus (SE). For reasons that are not fully understood, the efficacy of benzodiazepines decreases with increasing duration of seizure activity. This often forces clinicians to resort to more drastic second‐ and third‐line treatments that are not always successful. The antiseizure properties of benzodiazepines are mediated by γ‐aminobutyric acid type A (GABAA) receptors. Decades of research have focused on the failure of GABAergic inhibition after seizure onset as the likely cause of the development benzodiazepine resistance during SE. However, the details of the deficits in GABAA signaling are still largely unknown. Therefore, it is necessary to improve our understanding of the mechanisms of benzodiazepine resistance so that more effective strategies can be formulated. In this review we discuss evidence supporting the role of altered GABAA receptor function as the major underlying cause of benzodiazepine‐resistant SE in both humans and animal models. We specifically address the prevailing hypothesis, which is based on changes in the number and subtypes of GABAA receptors, as well as the potential influence of perturbed chloride homeostasis in the mature brain.

KCC2 activity is critical in limiting the onset and severity of status epilepticus

Significance Status epilepticus (SE) is defined as a state of continuous unremitting seizures that often exhibits underlying deficits in neuronal inhibition mediated by GABAA receptors. The efficacy of neuronal inhibition is critically dependent on the activity of the K+/Cl– cotransporter KCC2, which allows neurons to maintain low intracellular Cl– levels. KCC2 activity is enhanced by phosphorylation of residue serine 940, and here we show that SE leads to rapid dephosphorylation of this key regulatory residue. Moreover, we demonstrate that deficits in S940 phosphorylation directly contribute to the onset and severity of SE. Collectively, our results suggest that deficits in KCC2 activity directly contribute to the pathophysiology of SE. The K+/Cl– cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl– levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl– extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.

Selective Inhibition of KCC2 Leads to Hyperexcitability and Epileptiform Discharges in Hippocampal Slices and In Vivo

GABAA receptors form Cl− permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K+/Cl− cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl− levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in EGABA values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg2+ conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo.