Inflammatory mediator-induced modulation of GABA_A currents in human sensory neurons

Highlights

• Bicuculline-sensitive GABA_A currents are present in all human DRG neurons.

• The kinetics of GABA_A currents in rat DRG neurons are faster than those of human currents.

• The GABA current equilibrium potential was ∼20 mV more hyperpolarized than in rat neurons.

• Low- and high-affinity human GABA_A currents were increased by inflammatory mediators.

• Human sensory neurons may be a valuable tool to test compounds prior to use in humans.

Abstract

The purpose of the present study was to characterize the properties of A-type GABA receptor (GABA_A receptor) currents in human sensory neurons. Neurons were obtained from adult organ donors. GABA_A currents were recorded in isolated neurons. Both large inactivating low-affinity currents and smaller persistent high-affinity currents were present in all of the 129 neurons studied from 15 donors. The kinetics of human GABA_A currents were slower than those in rat sensory neurons. GABA currents were completely blocked by bicuculline (10 μM), and persistent currents were activated by the δ-subunit-preferring agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). The GABA current equilibrium potential was ∼20 mV more hyperpolarized than in rat neurons. Both low- and high-affinity currents were increased by inflammatory mediators but via different second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool to test compounds prior to use in humans.

Introduction

Behavioral pharmacological data from rodents indicate that A-type GABA receptor (GABA_A receptor) signaling is critically involved in the modulation of nociception at sites throughout the central nervous system (CNS) (Price et al., 2009). Particularly important is the gating of afferent input to the spinal cord. While data from rodent models also suggest that post-synaptic inhibitory circuitry in the spinal cord dorsal horn is likely to be important for regulation of nociceptive threshold, particularly in the presence of injury, available electrophysiological, pharmacological and morphological data suggest that pre-synaptic inhibition of afferent input is the dominant mechanism for inhibition of somatosensory input into the CNS (Eccles et al., 1962, Eccles et al., 1963, Nishi et al., 1974, Mokha et al., 1983, Hiura et al., 1998, Reeve et al., 1998, Rudomin and Schmidt, 1999, Bae et al., 2000, Olave et al., 2002, Sutherland et al., 2002, Sokal and Chapman, 2003, Vesselkin et al., 2003, Weng and Dougherty, 2005). Virtually all dorsal root ganglion (DRG) neurons from rat respond to GABA with a rapidly activating, bicuculline-sensitive anion current (Oyelese et al., 1995, Zhu et al., 2012a). However, in contrast to neurons in the CNS, activation of GABA_A receptors on primary afferents results in membrane depolarization. This paradoxical response is thought to be due to a relatively high concentration of intracellular Cl⁻ as a result of the persistent expression of the Na⁺–K⁺–Cl⁻-co-transporter, NKCC1 into adulthood.

Despite the wealth of knowledge of GABA_A signaling in rodents and the growing interest in the use of GABA_A receptor ligands for the treatment of pain, there is only one report of GABA_A currents in human sensory neurons (Maddox et al., 2004). Interestingly, the currents from adult human DRG neurons described in this study were resistant to the classical GABA_A receptor antagonists bicuculline and picrotoxin (Maddox et al., 2004). And while there appear to be limited differences between human and rodent GABA_A receptor homologs in heterologous expression systems, evidence of unique pharmacological properties of the GABA_A currents in human sensory neurons raises the possibility that the differences are due to processes unique to the native environment. Thus, given the therapeutic potential of GABA_A receptor ligands suggested by preclinical data (Witschi et al., 2011), and in light of growing concerns over the extent to which preclinical data translate to human clinical conditions (Seok et al., 2013), we sought to further characterize GABA_A currents in human sensory neurons.

Our results suggest that while there are many similarities between the GABA_A currents present in rodent and human sensory neurons, there are marked differences. These included biophysical properties of the evoked currents, which were more slowly activating and inactivating in human DRG neurons, as well as the response to inflammatory mediators. That is, in contrast to the selective potentiation of high-affinity GABA_A currents previously observed in rat DRG neurons (Lee and Gold, 2012), both low- and high-affinity currents were potentiated in human sensory neurons via what appeared to be distinct second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool with which to test compounds developed in heterologous expression systems and validated in rodent models prior to the use in humans.