The K+-Cl− cotransporter KCC2 is responsible for maintaining low Cl− concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABAA and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl− transport10, 11, 12. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl−]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl−]i. CLP257 restored impaired Cl− transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.
At a glance
- Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia. Arch. Gen. Psychiatry 68, 21–31 (2011). &
- Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury. Nat. Med. 16, 302–307 (2010). et al.
- Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424, 938–942 (2003). et al.
- Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis. Nat. Neurosci. 12, 438–443 (2009). , , &
- Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy. J. Neurosci. 27, 9866–9873 (2007). et al.
- Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia. J. Neurosci. 31, 11088–11095 (2011). et al.
- Role of cation-chloride-cotransporters (CCC) in pain and hyperalgesia. Curr. Top. Med. Chem. 5, 547–555 (2005). , &
- Behavioural phenotypes of hypomorphic KCC2-deficient mice. Eur. J. Neurosci. 21, 1327–1337 (2005). , , , &
- Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl− homeostasis. Nat. Neurosci. 16, 183–192 (2013). et al.
- Efficacy of synaptic inhibition depends on multiple, dynamically interacting mechanisms implicated in chloride homeostasis. PLoS Comput. Biol. 7, e1002149 (2011). et al.
- Altered chloride homeostasis in neurological disorders: a new target. Curr. Opin. Pharmacol. 7, 93–99 (2007).
- Roles of the cation-chloride cotransporters in neurological disease. Nat. Clin. Pract. Neurol. 4, 490–503 (2008). et al.
- A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27, 447–459 (2000). &
- The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2. Cell Physiol. Biochem. 22, 31–44 (2008). &
- Brain-type creatine kinase activates neuron-specific K+-Cl− co-transporter KCC2. J. Neurochem. 96, 598–608 (2006). , , &
- Homooligomeric and heterooligomeric associations between K+-Cl− cotransporter isoforms and between K+-Cl− and Na+-K+-Cl− cotransporters. J. Biol. Chem. 282, 18083–18093 (2007). et al.
- Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001). , , &
- Cloning, characterization, and chromosomal location of a novel human K+-Cl− cotransporter. J. Biol. Chem. 274, 10661–10667 (1999). et al.
- Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2. Proc. Natl. Acad. Sci. USA 106, 5383–5388 (2009). et al.
- Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: ultrastructural morphometric analysis of axonal alterations. Pain 64, 37–57 (1996). &
- BDNF-induced TrkB activation down-regulates the K+-Cl− cotransporter KCC2 and impairs neuronal Cl− extrusion. J. Cell Biol. 159, 747–752 (2002). et al.
- Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J. Neurosci. 24, 4683–4691 (2004). et al.
- BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017–1021 (2005). et al.
- Reduced potassium-chloride co-transporter expression in spinal cord dorsal horn neurons contributes to inflammatory pain hypersensitivity in rats. Neuroscience 152, 502–510 (2008). , &
- Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat. J. Physiol. (Lond.) 586, 5701–5715 (2008). , , , &
- The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury. Mol. Pain 4, 36 (2008). et al.
- Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters. Pain 140, 48–57 (2008). , , &
- Nerve constriction in the rat: model of neuropathic, surgical and central pain. Pain 83, 37–46 (1999). , &
- A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107 (1988). &
- Gabapentin and pregabalin for chronic neuropathic and early postsurgical pain: current evidence and future directions. Curr. Opin. Anaesthesiol. 20, 456–472 (2007).
- Activation of 5–HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2. Proc. Natl. Acad. Sci. USA 110, 348–353 (2013). et al.
- 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 104, 933–946 (2001). et al.
- Expression of the neuron-specific potassium chloride cotransporter KCC2 in adult rat cochlear. Neurosci. Lett. 441, 205–209 (2008). , , , &
- Electroneutral cation-chloride cotransporters in the central nervous system. Neurochem. Res. 29, 17–25 (2004). , &
- The neuropathic pain market. Nat. Rev. Drug Discov. 11, 101–102 (2012).
- Antiepileptic drugs for the treatment of neuropathic pain: a systematic review. Med. Oral Patol. Oral Cir. Bucal 17, e786–e793 (2012). , , &
- Efficacy of antidepressants as analgesics: a review. J. Clin. Pharmacol. 52, 6–17 (2012). , &
- Opioids and neuropathic pain. Pain Physician 15, ES93–ES110 (2012).
- A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association. J. Neurosci. 25, 6971–6983 (2005). et al.
- Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor. J. Neurosci. 31, 12916–12926 (2011). et al.
- The phasor approach to fluorescence lifetime imaging analysis. Biophys. J. 94, L14–L16 (2008). , , &
- Pharmacological enhancement of δ-subunit–containing GABAA receptors that generate a tonic inhibitory conductance in spinal neurons attenuates acute nociception in mice. Pain 152, 1317–1326 (2011). et al.
- Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity. J. Neurosci. 25, 9613–9623 (2005). , , , &
- Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn. Mol. Pain 5, 24 (2009). , , , &
- Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63 (1994). , , , &
- Supplementary Text and Figures (142 KB)
Supplementary Tables 1–3 and Supplementary Figure 1