Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K⁺ Channel Dysfunction in DRG Neurons

Abstract

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI. Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1).

A-Type KV Channels in Dorsal Root Ganglion Neurons: Diversity, Function, and Dysfunction

“…Kv3.4 immunoreactivity in the superficial rat spinal dorsal horn provided strong evidence for the expression of Kv3.4 channels in DRG nociceptors (Brooke et al, 2004 ; Chien et al, 2007 ; Muqeem et al, 2018 ). Specifically, the axon, soma and presynaptic terminals of rat DRG neurons demonstrated significant Kv3.4 immunoreactivity (Figure 3 ; Chien et al, 2007 ; Ritter et al, 2015a ; Zemel et al, 2017 ; Muqeem et al, 2018 ). In the somata of DRG neurons, Kv3.4 channels are found in all neurons, although it appears to be especially enriched in small-diameter neurons (Chien et al, 2007 ; Ritter et al, 2012 , 2015a ; Zemel et al, 2017 ).…”

“…Specifically, the axon, soma and presynaptic terminals of rat DRG neurons demonstrated significant Kv3.4 immunoreactivity (Figure 3 ; Chien et al, 2007 ; Ritter et al, 2015a ; Zemel et al, 2017 ; Muqeem et al, 2018 ). In the somata of DRG neurons, Kv3.4 channels are found in all neurons, although it appears to be especially enriched in small-diameter neurons (Chien et al, 2007 ; Ritter et al, 2012 , 2015a ; Zemel et al, 2017 ). The immunoreactivities of Kv3.4, Nav1.8, and TRPV1 colocalize, which is consistent with expression in nociceptors (Chien et al, 2007 ; Ritter et al, 2015a ).…”

“…Although PKC was known to phosphorylate Kv3.4 channels causing an acute loss of inactivation, nothing was known about the phosphatases that countered PKC activity. We found that pharmacological inhibition of CaN was sufficient to not only slow inactivation, but also attenuate Kv3.4 currents (Zemel et al, 2017 ). These modulations are dependent on the presence of the NTID PKC sites at S8, S9, S15, and S21 (Zemel et al, 2017 ).…”

“…In DRG neurons, the modulation by G-protein coupled receptors occurs by a membrane-delimited mechanism suggesting the presence of a Kv3.4 channel-receptor-PKC complex (Ritter et al, 2012 ). The phosphatase calcineurin (CaN) seemingly opposes the activity of PKC as inhibiting CaN with small molecules or overexpressing the regulator of calcineurin 1 (RCAN1) reduces Kv3.4 inactivation (Zemel et al, 2017 ). High levels of phosphatidylinositol 4,5-bisphosphate (PIP2) may also alter Kv3.4 channel inactivation but this has not been confirmed in a physiological setting (Oliver et al, 2004 ; Kruse et al, 2012 ).…”

“…Following implantation of a bone tumor near the sciatic nerve, immunoreactivity of DRG Kv3.4 channels was reduced as determined by western blot (Duan et al, 2012 ). Finally, in a model of unilateral spinal cord contusion, Kv3.4 current amplitude, inactivation, and channel membrane expression are reduced in the DRG (Ritter et al, 2015a ; Zemel et al, 2017 ). Western blot and single-cell quantitative PCR results indicate that total protein and mRNA in the DRG had not changed, suggesting a possible post-translational effect (Ritter et al, 2015a ).…”