MrgD activation inhibits KCNQ/M-currents and contributes to enhanced neuronal excitability

Abstract

The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, beta-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of G(i/o) and phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, beta-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of beta-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before beta-alanine application fired multiple APs during beta-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.

Figure 1.

β-Alanine-induced activation of MrgD strongly inhibits KCNQ2/3 currents in CHO cells. A, Typical step depolarization voltage protocol used to elicit activation of KCNQ2/3 currents. B, Sample sweeps before (1) and ∼1 min into (2) β-alanine (100 μm) exposure. C, Concentration–response relationship for 1, 10, 100, and 500 μm β-alanine. D, Time course of MrgD modulation of KCNQ2/3 currents. Currents were maximally inhibited within ∼1 min of exposure to β-alanine (100 μm) but only when MrgD was present. 1 and 2 refer to times when currents were averaged and displayed in B. Open symbols, Plus MrgD; closed symbols, minus MrgD; open horizontal bar, duration of β-alanine exposure.

Figure 2.

Differential coupling of KCNQ2/3 and M-channels to MrgD-activated G-proteins in CHO cells and DRG neurons, respectively. A, Whole-cell voltage-clamp experiments revealed that MrgD activation by β-alanine (100 μm) in CHO cells containing KCNQ2/3 resulted in strong suppression of evoked currents. This effect was partially blocked by PTX treatment (100 ng/ml; overnight) and completely prevented by acute inhibition of phospholipase C (U73122, 1 μm; edelfosine, 10 μm). *Group is statistically larger than all other groups (p < 0.001). ^#Statistically smaller than β-alanine control response and larger than the PLC inhibitor groups (p < 0.017). B, In contrast, M-currents in DRG neurons were only partially blocked by PLC inhibition, but responses to β-alanine were completely prevented by PTX treatment (100 ng/ml; overnight). *Group is statistically larger than all other groups (p < 0.001). ^#Statistically smaller than β-alanine control response and larger than PTX group (p < 0.017). Ctrl, Control.

Figure 3.

Activation of endogenous MrgD suppresses M-currents and enhances phasic DRG neuron excitability. A, Top depicts the voltage protocol used to monitor M-currents. Sample sweeps during baseline and β-alanine exposure (500 μm) illustrate MrgD-induced suppression of M-currents. For summary data and effects of G-protein signaling inhibitors, see Figure 2 B. The amplitude of M-currents was measured as the difference in mean current indicated by the brackets between the onset (∼10 ms) and end of the hyperpolarizing pulse. B, A consequence of β-alanine exposure is enhanced neuronal excitability reflected as an increase in action potential firing rate.