Invited reviewCation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments
Highlights
► Cation-chloride cotransporters (CCCs) modulate the efficacy of GABAergic inhibition. ► Two members of the CCC family, KCC2 and NKCC1, are of particular importance. ► Both transporters are potential targets for novel antiepileptic and antiepileptogenic treatments. ► The NKCC1 inhibitor bumetanide has been widely studied in this respect. ► This review critically discusses the pros and cons of CCC blockers for epilepsy therapy.
Introduction
Epilepsy, one of the most common disorders of the brain, is characterized by recurrent, usually unprovoked epileptic seizures, and there is a wide spectrum of cognitive, psychosocial, and social consequences of this condition. Ictogenesis, the rapid process of initiation and propagation of a seizure in time and space, is symptomatic of the underlying pathology.
A variety of genetic and developmental abnormalities as well as brain insults, including traumatic brain injury, ischemic stroke, intracerebral hemorrhage, status epilepticus (SE) and encephalitis, have the potential to induce the development of epilepsy in humans and rodent disease models (Cepeda et al., 2006; Chang and Lowenstein, 2003; Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009). The mechanisms underlying this gradual process, termed epileptogenesis, whereby brain is altered becoming susceptible to spontaneous recurrent seizures, are only incompletely understood, but they include inflammation, neurodegeneration, blood–brain barrier (BBB) disruption, and alterations in expression and function of diverse receptors and ion channels (Dichter, 2009; Heron et al., 2007; Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009; Timofeev et al., 2010).
During the recent years, changes in plasmalemmal ion-transport mechanisms, which are instrumental for ion channel function and volume regulation, have been suggested to play pivotal roles in epileptic processes (Blaesse et al., 2009).
Seizures occur more often during the neonatal period than at any other age (Annegers et al., 1995; Hauser et al., 1993). Neonatal seizures are deleterious events and they can have profound long-term consequences, often leading to chronic epilepsy and significant cognitive and motor disabilities (Rakhade and Jensen, 2009). A well-recognized, major clinical problem is that neonatal epileptic seizure activity shows only limited response to commonly used GABA-mimetic and GABA-modulating antiepileptic drugs (AEDs; also known as anticonvulsant, anti-ictal or anti-seizure drugs) such as phenobarbital and benzodiazepines (Bonifacio et al., 2011; Painter et al., 1999; Rennie and Boylan, 2007), which enhance the inhibitory actions mediated by GABAA receptors in adults (Rogawski and Löscher, 2004). AEDs used in the treatment of epilepsy in adults are often ineffective in neonates and can even potentiate seizure episodes. Hence, the search for novel AEDs and other therapeutic strategies (Helmy et al., 2011) is particularly important in the case of neonatal seizures. In fact, with regard to neonatal and pediatric seizures, controlling neuronal pHi might turn out to be a very successful way to manipulate neuronal excitability (Helmy et al., 2011; Schuchmann et al., 2006). A decrease in pHi of 0.05 units was induced in a quantitatively identical manner by various membrane-permeant weak organic acids as well as isohydric hypercapnia, and the intraneuronal acidosis had a pronounced suppressing action on neuronal network activity (Ruusuvuori et al., 2010). This effect has nothing to do with mitochondrial energy metabolism (see Holmgren et al., 2010; and Bregestovski and Bernard, 2012) as is evident from the fact that quantitatively similar suppression of giant depolarizing potential (GDP) generation took place regardless of whether the weak acid applied was (l-lactate) or was not (d-lactate, propionate) an effective substrate of mitochondrial ATP production (Ruusuvuori et al., 2010). Moreover, CO2 is an end-product of energy metabolism. Mitochondrial membrane potential within the neurons was not affected by any of these weak acids, but a strong dependence on the availability of glucose was evident (Ruusuvuori et al., 2010).
About 30% of adult patients with epilepsy do not respond to currently used AEDs. Such AED resistance is associated with serious comorbidity and increased mortality, warranting an urgent need for more effective AEDs (Löscher and Schmidt, 2011). In addition, prevention of epilepsy in patients at risk after brain insults remains an unmet medical need, therefore, development of drugs that target the mechanisms of epileptogenesis is important (Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009). A modest acid load has been shown to be effective in suppressing drug-resistant seizures in adults (Tolner et al., 2011). However, we will focus below on drugs acting on Cl− regulation in the control of neonatal, pediatric and adult seizures.
Recently, inhibitors of plasmalemmal cation-chloride cotransporters (CCCs) such as furosemide and, especially, bumetanide have attracted much interest as putative AEDs as will be described in detail in this review. Considering the multitude of overlapping mechanisms and endpoints of major central nervous system (CNS) diseases, it is perhaps not surprising that the above drugs have been also implicated in the treatment of conditions such as cerebral edema and swelling-related neurodegeneration after ischemic and traumatic brain injury (Kahle et al., 2008; Kintner et al., 2007; Walcott et al., 2012), chronic pain (Price et al., 2005), as well as autism (Lemonnier and Ben-Ari, 2010) and conditioned anxiety (Krystal et al., 2012).
Section snippets
Physiology and pathophysiology of cation-chloride cotransporters in the brain
The electrochemical gradient of Cl− across the neuronal plasma membrane is controlled by CCCs (for review, see Blaesse et al., 2009). CCCs are secondary active transporters that drive net Cl− extrusion (K–Cl cotransporters, KCCs) or uptake (Na–K–2Cl cotransporters, NKCCs) by using the K+ and Na+ gradients which, in turn, are generated by the Na–K ATPase (the “sodium pump”). The neuron-specific KCC isoform, KCC2, is the main Cl− extruding mechanism in hippocampal and neocortical principal
Basic mechanisms of GABAergic inhibition: hyperpolarization and shunting
GABAA-mediated transmission is not globally suppressed in epileptic tissue (Avoli et al., 2005; Avoli and de Curtis, 2011; Köhling et al., 1998; Mann and Mody, 2008), but the neuronal Cl− extrusion capacity, which is an important factor in controlling the efficacy of postsynaptic inhibition (see below) may play a role in setting the susceptibility of neurons to epileptiform activity, at least under certain conditions (Ben-Ari et al., 2007; Blaesse et al., 2009; Briggs and Galanopoulou, 2011).
Pharmacological targeting of NKCC1 and KCC2
Based on the assumption that abnormal functional expression of KCC2 and NKCC1 might promote ictal activity (Kahle et al., 2008), these Cl− transporters have been suggested as targets for antiepileptic and antiepileptogenic treatments. As will be explained below, pharmacological inhibition of not only NKCC1 but also of KCC2 in central neurons might have antiepileptic effects.
Epileptogenesis in neonates
Most of the available data does not suggest that GABA would be overtly depolarizing in the healthy newborn human cortex (Section 2.). However, the situation might be different in preterm babies as well as in full term neonates with epilepsy. Neonatal epilepsy is often caused by birth trauma, such as asphyxia or hemorrhages (Ronen et al., 1999), which is expected to lead to neuronal damage and de-differentiation and, consequently, to a high neuronal [Cl−]i.
Recent experimental work by the groups
Ictogenesis in neonates
As discussed above, the depolarizing actions of GABA in immature neurons are a consequence of the high [Cl−]i. In theory, inhibition of NKCC1, by reducing [Cl−]i, could reduce GABA-mediated excitation or even potentiate the well-known inhibitory modes of GABA signaling in neonatal neurons (see Section 3.; and Lamsa et al., 2000; Wells et al., 2000).
Dzhala et al. (2005) reported that bumetanide (0.1–0.2 mg/kg i.p.) attenuated kainate-induced electrographic seizures in neonatal rats (but see
Conclusions
The work on the antiepileptic and antiepileptogenic effects of NKCC1 and KCC2 inhibitors have revealed at least four distinct mechanisms of action which may or may not show overlap under various experimental and clinical conditions.
- (1)
The most common working hypothesis in studies of diuretic agents on epilepsy posits that a major epilepsy-promoting mechanism is an NKCC1-mediated increase in neuronal [Cl−i] which diminishes the efficacy of GABAergic inhibition or even reverts it to excitation. A
Acknowledgments
We thank Daryl Hochman for helpful discussion and for providing details on his monkey experiments, as well as Peter Blaesse, Mohamed Helmy and Eva Ruusuvuori for comments on an early version of this paper.
The authors' original research work was supported by grants from the Letten Foundation, the Academy of Finland, the Sigrid Juselius Foundation, the Jane and Aatos Erkko Foundation (K.K.) and the Deutsche Forschungsgemeinschaft (Bonn, Germany; Lo 274/11; W.L.). K.K. is a member of the Finnish
References (183)
- et al.
Na, K-ATPase is decreased in hippocampus of kainate-lesioned rats
Epilepsy Res.
(1994) Seizure and acute osmotic change: clinical and neurophysiological aspects
J. Neurol. Sci.
(1991)- et al.
GABAergic synchronization in the limbic system and its role in the generation of epileptiform activity
Prog. Neurobiol.
(2011) - et al.
Cellular and molecular mechanisms of epilepsy in the human brain
Prog. Neurobiol.
(2005) - et al.
Effect of mannitol treatment on brain neurotransmitter markers in kainic acid-induced epilepsy
Neuroscience
(1987) - et al.
Cation-chloride cotransporters and neuronal function
Neuron
(2009) - et al.
Epileptogenesis in pediatric cortical dysplasia: the dysmature cerebral developmental hypothesis
Epilepsy Behav.
(2006) - et al.
Translating developmental time across mammalian species
Neuroscience
(2001) - et al.
Osmolality-induced changes in extracellular volume alter epileptiform bursts independent of chemical synapses in the rat: importance of non-synaptic mechanisms in hippocampal epileptogenesis
Neurosci. Lett.
(1990) - et al.
The cellular, molecular and ionic basis of GABAA receptor signalling
Prog. Brain Res.
(2007)
Na+K+ ATPase activity in the rat hippocampus: a study in the pilocarpine model of epilepsy
Neurochem. Int.
[3H]bumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na, K, Cl co-transport system
J. Biol. Chem.
Antiepileptic effect of loop diuretics? Comment to “Differential effects of cation-chloride co-transport-blocking diuretics in a rat hippocampal slice model of epilepsy” by Margineanu, Klitgaard, Epilepsy Res. 69 (2006) 93–99
Epilepsy Res
Cetyl GABA: effect on convulsant thresholds in mice and acute toxicity
Neuropharmacology
Influence of inhibitors of the high affinity GABA uptake on seizure thresholds in mice
Neuropharmacology
Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium–(potassium)–chloride cotransporter family expressed in kidney
J. Biol. Chem.
Cyclicity of spontaneous recurrent seizures in pilocarpine model of temporal lobe epilepsy in rat
Exp. Neurol.
Diuretic complications
Am. J. Med. Sci.
Channelopathies in idiopathic epilepsy
Neurotherapeutics
Expression of the Na–K–2Cl-cotransporter NKCC1 during mouse development
Mech. Dev.
Disruption of KCC2 reveals an essential role of K–Cl cotransport already in early synaptic inhibition
Neuron
Ionic basis of GABA(A) receptor channel function in the nervous system
Prog. Neurobiol.
The differential expression patterns of messenger RNAs encoding K–Cl cotransporters (KCC1,2) and Na–K–2Cl cotransporter (NKCC1) in the rat nervous system
Neuroscience
Long-term effectiveness and side effects of acetazolamide as an adjunct to other anticonvulsants in the treatment of refractory epilepsies
Brain Dev.
Model-specific effects of bumetanide on epileptiform activity in the in-vitro intact hippocampus of the newborn mouse
Neuropharmacology
Mouse organic anion transporter 2 and 3 (mOAT2/3[Slc22a7/8]) mediates the renal transport of bumetanide
Eur. J. Pharmacol.
Tyrosine phosphorylation regulates the membrane trafficking of the potassium chloride co-transporter KCC2
Mol. Cell Neurosci.
Inhibition of sodium–potassium-ATPase: a potentially ubiquitous mechanism contributing to central nervous system neuropathology
Brain Res. Brain Res. Rev.
Controlled trial of frusemide as an antiepileptic drug in focal epilepsy
Br. J. Clin. Pharmacol.
The molecular basis of water transport in the brain
Nat. Rev. Neurosci.
Mechanisms of tolerance to the anticonvulsant effects of acetazolamide in mice: relation to the activity and amount of carbonic anhydrase in brain
Epilepsia
Secular trends and birth cohort effects in unprovoked seizures: Rochester, Minnesota 1935–1984
Epilepsia
Positive shifts of the GABAA receptor reversal potential due to altered chloride homeostasis is widespread after status epilepticus
Epilepsia
A molecular neuroanatomical study of the developing human neocortex from 8 to 17 postconceptional weeks revealing the early differentiation of the subplate and subventricular zone
Cereb. Cortex
Giant synaptic potentials in immature rat CA3 hippocampal neurones
J. Physiol.
GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations
Physiol. Rev.
Is it safe to use a diuretic to treat seizures early in development?
Epilepsy Curr.
Membrane physiology–bridging the gap between medical disciplines
N. Engl. J. Med.
Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations
N. Engl. J. Med.
A new neurological focus in neonatal intensive care
Nat. Rev. Neurol.
Development of epileptiform excitability in the deep entorhinal cortex after status epilepticus
Eur. J. Neurosci.
Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy
J. Neurosci.
Clinical pharmacology of loop diuretics in health and disease
Eur. Heart J.
Excitatory GABA: how a correct observation may turn out to be an experimental artifact
Front. Pharmacol.
Altered GABA signaling in early life epilepsies
Neural Plast.
Thermodynamic regulation of NKCC1-mediated Cl- cotransport underlies plasticity of GABAA signaling in neonatal neurons
J. Neurosci.
Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study
J. Physiol.
Sensors and regulators of intracellular pH
Nat. Rev. Mol. Cell Biol.
A claim for caution in the use of promising bumetanide to treat neonatal seizures
J. Child. Neurol.
Epilepsy
N. Engl. J. Med.
Cited by (211)
Low-frequency stimulation in the zona incerta attenuates seizure via driving GABAergic neuronal activity
2024, Neurobiology of DiseaseFunctional interactions between potassium-chloride cotransporter (KCC) and inward rectifier potassium (Kir) channels in the insect central nervous system
2023, Pesticide Biochemistry and PhysiologyAltered childhood brain development in autism and epilepsy
2023, Encyclopedia of Child and Adolescent Health, First Edition