The WNK1/HSN2 kinase, mutated in a hereditary form of pain insensitivity, contributes to neuropathic pain and is a potential new target for analgesic drugs, reports a new mouse study. Using a WNK1/HSN2 knockout mouse, researchers led by Kristopher Kahle, now at Yale School of Medicine, New Haven, US, and Guy Rouleau, McGill University, Montreal, Canada, demonstrate that WNK1/HSN2 increases the phosphorylation of the potassium-chloride co-transporter KCC2, leading to a maladaptive decrease in KCC2 function and a loss of γ-aminobutyric acid (GABA)-mediated inhibition of pain-sensing neurons in the spinal cord. They further show that inhibiting the kinase lessens neuropathic pain in mice by restoring GABA inhibition.
The new findings were published March 29 in Science Signaling.
“This is another nice illustration that the KCC2 mechanism is involved in neuropathic pain. The most important lesson from this paper is the demonstration that it’s possible to rescue KCC2 function as a means of producing analgesia in neuropathic pain,” said Yves De Koninck, Laval University, Quebec City, Canada, who was not involved in the new study. In previous work, De Koninck and colleagues showed that a compound that enhanced KCC2 activity improved neuropathic pain in a rat model (see PRF related news story).
From the clinic to an animal study
In previous work, Rouleau and colleagues discovered that mutations in HSN2, a nervous system-specific exon of the gene that makes the WNK1 kinase, lead to hereditary sensory and autonomic neuropathy type IIA (HSANII), an autosomal recessive inherited form of congenital pain insensitivity (Shekarabi et al., 2008).
However, how disruption of this kinase produced the pain insensitivity was unknown, said Kahle. To explore further, co-first authors Kahle, Jean-François Schmouth, and Valérie Lavastre used Cre recombinase technology to create a knockout mouse lacking the HSN2 exon of WNK1 (WNK1/HSN2 knockouts).
Overall, the knockouts appeared mostly normal. Spinal and sensory nerve morphology was intact, and a battery of general behavioral and neurological tests revealed no abnormalities. The researchers did find a significant increase in the latency of male knockouts’ tail withdrawal from 47°C and 49°C water, but observed no other differences from wild-type animals in other tests of noxious thermal, mechanical, or chemical sensitivity.
"Knocking out the whole gene didn't have a very big effect," Kahle told PRF. "However, digging a little deeper, we wondered if maybe the kinase is protective or exacerbative in conditions under which we know this pathway might be important, such as neuropathic pain,” he added.
Consequently, the researchers next examined the effect of knocking out the gene in the spared nerve injury (SNI) model of neuropathic pain and in the complete Freund’s adjuvant (CFA) model of inflammatory pain. Compared to their wild-type counterparts, SNI-treated WNK1/HSN2 knockout mice displayed significantly reduced levels of cold hyperalgesia and mechanical allodynia. In contrast, both wild-type animals and the knockouts displayed similar levels of mechanical and thermal nociception in the CFA model. Together, the results suggest that WNK1/HSN2 knockouts show reduced pain hypersensitivity following nerve injury, but not following inflammation.
“Taken in the context of the finding that complete knockout of the protein didn’t have any bad systemic effects, our results are exciting and suggest that inhibition of the WNK1/HSN2 kinase with a drug would not only produce no side effects, but would selectively impact neuropathic pain behaviors without toxicity,” Kahle said.
What’s the mechanism?
The researchers next turned to the mechanism underlying the diminished pain in the knockouts. Based on evidence that a kidney-specific isoform of WNK1/HSN2 regulates a sodium-chloride co-transporter (called NCC) to control blood pressure, the researchers hypothesized that the nervous system-specific isoform of WNK1/HSN2 might act similarly to regulate the phosphorylation of KCC2, which is related to NCC and has long been known to play a role in neuropathic pain.
“Numerous groups have shown before that KCC2’s function is inhibited or completely reduced in neuropathic pain, but no one really knew why,” Kahle explained.
KCC2, present on dorsal horn neurons and across the central nervous system, pumps chloride out of cells. This activity helps generate a chloride gradient that affects GABA signaling onto postsynaptic dorsal horn neurons; GABA is the main inhibitory neurotransmitter in the central nervous system. When KCC2 is functioning properly to keep intracellular chloride concentrations low, GABA has a hyperpolarizing, inhibitory effect on neurons, Kahle told PRF. In contrast, when intracellular chloride concentrations are high, as occurs with deficient KCC2 activity, GABA has a depolarizing, excitatory effect.
The phosphorylation status of KCC2 by WNK1/HSN2 regulates the co-transporter’s activity. When specific threonine (Thr) amino acid residues are not phosphorylated, KCC2 is active, leading to increased chloride extrusion from the cell and an inhibitory effect of GABA. In contrast, phosphorylation at these same residues inhibits KCC2 activity, decreases chloride extrusion, and results in the loss of GABA inhibition.
To take a closer look at the phosphorylation status of KCC2 in the knockouts, the researchers examined two Thr residues in the co-transporter, looking at the spinal cord of naïve and SNI mice. In naïve WNK1/HSN2 knockout mice, phosphorylation levels of the Thr residues were significantly lower compared to those of naive mice with intact Wnk1. SNI increased phosphorylation of KCC2 at the Thr residues in both genotypes, but the amount of phosphorylation in the knockout mice was equal to that of naïve mice with intact Wnk1. The authors concluded that the WNK1/HSN2 kinase decreases the activity of KCC2 by increasing the inhibitory phosphorylation of the co-transporter at the Thr residues in the SNI model.
Finally, electrophysiological experiments in spinal cord slices showed that the WNK1/HSN2 kinase alters chloride levels so that GABA signaling increases neuronal activity rather than decreases it; it is this reversal of GABA’s normally inhibitory effect on neurons that presumably results in neuropathic pain. The group then demonstrated that antagonism of the kinase, achieved through WNK1/HSN2 knockout or through use of a pharmacological inhibitor, restored GABA’s inhibitory role in spinal cord neurons from SNI animals.
“We showed that the reason that KCC2 is inhibited in the SNI model of neuropathic pain is because of excessive maladaptive phosphorylation at two threonine residues by WNK1/HSN2,” said Kahle. “By inhibiting WNK1/HSN2, whether through genetics or drugs, we reduced the maladaptive phosphorylation, restored chloride gradients, and restored GABA inhibition, so that [presumably] there is no excessive pain signal sent to the central nervous system in neuropathic pain,” he continued. The current study provides the motivation for examining in more detail the clinical utility of inhibiting the WNK1 pathway as a treatment for neuropathic pain, and the team is currently working on developing new WNK1 inhibitors, he added.
“This study is yet another piece of evidence suggesting that KCC2 is an important player to target for analgesic drug discovery,” said De Koninck. Loss of KCC2 function has been associated with several neurological disorders besides chronic pain, including epilepsy, motor spasticity, stress disorders, and possibly schizophrenia. “It will be intriguing to know if targeting KCC2 may also [prove to be a] therapeutic avenue for these other disorders,” he said.
Image credit: Kahle et al., reproduced with permission of the American Association for the Advancement of Science