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

Abstract

The purpose of the present study was to characterize the properties of A-type GABA receptor (GABAA receptor) currents in human sensory neurons. Neurons were obtained from adult organ donors. GABAA 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 GABAA 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.

Keywords: dorsal root ganglion; intracellular Cl(−) concentration; patch clamp; protein kinase A; protein kinase C; tyrosine kinase.

Figure 1

High and low affinity GABA_A currents in human sensory neurons. A. In acutely dissociated neurons, a low affinity transient current predominates. GABA was applied at the time indicated. B. After 24hr or more in culture, a high affinity persistent current was detectable. C. Concentration-response data from 10 neurons were normalized with respect to the amplitude of the current evoked with 1 mM GABA. Pooled data were fitted with a Hill equation to yield an EC50 of 540 nM.

Figure 2

The kinetics of low affinity GABA (1 mM) evoked currents in human sensory neurons are slower than those observed in rat sensory neurons. A. The fast component of current inactivation was well fitted with a single exponential equation (gray trace) used to estimate the inactivation time constant. Inset: A single exponential function was also used to determine the time constant of current activation. B. Pooled inactivation time constant (tau) data from 11 human and 9 rat neurons were analyzed with a t-test which confirmed that the difference between groups was significant. C. Pooled activation time constant (tau) data from human and rat neurons was also analyzed with a t-test confirming that activation of GABA_A currents in rat sensory neurons was faster than in human sensory neurons. * is p < 0.05 and ** is p < 0.01.

Figure 3

GABA_A currents from adult human sensory neurons were blocked by bicuculline. GABA was applied as indicated alone (black trace) and then 5 minutes later in combination with bicuculline (gray trace). Both low (A) and high (B) affinity currents were completely blocked by bicuculline. Comparable results were obtained in all 5 of the other neurons tested.

Figure 4

The GABA_A current equilibrium potential in human sensory neurons is depolarized relative to normal resting membrane potentials. A. A ramp protocol was used to determine the equilibrium potential for the GABA evoked currents. A step-and ramp voltage clamp protocol (bottom trace) was used to evoke current in the presence (gray trace) and absence (black trace) of GABA (applied half way through the step to −30 mV). B. The difference current was plotted relative to the ramp voltage, and the point of 0 current was considered the equilibrium potential. The mean (± SEM) of the equilibrium potential determined from 6 neurons from two different donors is indicated.

Figure 5

High and low affinity GABA_A currents in human sensory neurons are modulated by inflammatory mediators. A. GABA was applied before and then 3 minutes after the application of a combination of inflammatory mediators (IM). In the majority of neurons tested (15 of 16), the high affinity current (evoked with 10 µM) GABA was increased following IM application. B. Changes in low affinity currents were studied in the same neurons, and in half of these (6 of 13), the low affinity current was also increased by IM. Inset: In a minority of neurons (3 of 13), however, a decrease in low-affinity current was observed. Separate groups of neurons were pretreated with the TK kinase inhibitor, genistein (Gen, n = 11), the PKA inhibitor H-89 (n = 8), or the PKC inhibitor BIM (n = 5) prior to the application of GABA and then GABA with inflammatory mediators. Pooled high (C) and low (D) affinity current data were analyzed as a percent change from the baseline GABA current evoked from neurons treated with IM alone or pre-treated with kinase inhibitors. A two-way ANOVA indicated a significant interaction between current type (high vs low affinity) and treatment. Of note, only the neurons in which low affinity current increased by > 10% were included in this analysis, and plotted in panel D. The IM-induced decrease in low affinity current (D inset) was observed in a subpopulation of neurons pre-incubated with each inhibitor and no significant impact of inhibitor was detected in these neurons. Finally, a group of neurons (n = 5) was treated with PGE₂ (P) alone, rather than in combination with bradykinin and histamine. Pooled high (C) and low (D) affinity current data were included with plots of pooled IM data for comparison.