Nociceptin inhibits T-type Ca2+ channel current in rat sensory neurons by a G-protein-independent mechanism

Abstract

Nociceptin (orphanin FQ) is a novel, opioid-like, heptadecapeptide that is an endogenous ligand for the opioid receptor-like (ORL1) receptor. Unlike classical opioids, nociceptin can produce hyperalgesia when injected intracerebroventricularly into mice. Despite this, nociceptin has been reported to decrease transmitter release, activate an inwardly rectifying K+ conductance, and suppress high-voltage-activated Ca2+ channel conductances (HVA gCa) in much the same way as micro-, delta-, and kappa-opioids. We report an action of nociceptin that is not shared by morphine: the suppression of low-voltage-activated, transient calcium (barium) current (IBa,T) in acutely dissociated rat dorsal root ganglion (DRG) neurons (EC50 = 100 nM). This effect was reflected as inhibition of bursts of action potentials that can be evoked in "medium-sized" DRG neurons. Experiments with GTP-gamma-S (100 microM), GDP-beta-S (2 mM), or aluminum fluoride (AlF3) (100 microM) in the patch pipette failed to provide evidence for G-protein involvement in nociceptin-induced IBa,T suppression. By contrast, both morphine and nociceptin suppressed HVA gCa, and the latter response was affected by intracellular GTP-gamma-S, GDP-beta-S, and AlF3 in ways that confirmed G-protein involvement. The selective effect of nociceptin on IBa,T may therefore be relevant to understanding why its behavioral actions differ from those of other opioids. This G-protein-independent effect of the action of nociceptin may reflect a new general mechanism of action for opioid peptides within the nervous system.

Fig. 6.

Biophysical aspects of nociceptin-induced T-current suppression. A, Slow speed chart record to show rate of onset and the effect of nociceptin with and without repeated activation of g_Ba,T. Top record is current, and bottom record is voltage. Deflections result from brief (30 msec) voltage commands to −40 mV from the holding potential of −90 mV applied once every 2.5 sec (0.4 Hz). Nociceptin (1 μm) was first applied (atleft) while g_Ba,T was repeatedly activated. The current deflections attain a smaller steady-state amplitude 3 min after initiating nociceptin superfusion. Nociceptin was reapplied (at right) in the absence ofg_Ba,T activation. Voltage commands were recommenced 3 min after initiating nociceptin superfusion, andI_Ba,T responses were attenuated to the same extent as those recorded when the conductance had been activated repetitively in the presence of nociceptin. B, Normalized steady-state inactivation plots forg_Ba,T acquired before and during application of 100 nm nociceptin. Cell was held at a series of prepulse potentials (+5 mV increments from −115 mV) for 200 msec before activating g_Ba,T at −40 mV. Voltage protocol is shown as inset.C₁,C₂, Original data records used to produce the data in B. A split-clock protocol was used so that 100 msec calibration refers to prepulse and 25 msec calibration refers to recordings of I_Ba,T.

Fig. 1.

Blockade of I_Ba,T by nociceptin. A₁, A₂, Comparison of the effects of nociceptin and morphine onI_Ba,T in the same neuron. Voltage record (step to −40 mV from a holding potential of −90 mV) omitted for clarity. Large capacity current transients accompanying voltage steps have been attenuated or removed by erasing three or more data points (clock speed for data acquisition was 0.05 msec/point).A₁, Superimposed records to show almost complete suppression of I_Ba,T by 1 μmnociceptin. A₂, In the same cell, 1 μm morphine suppresses the current by <10%.B, Superimposed records obtained from another cell to show suppression of I_Ba,T tails by 1 μm nociceptin. Tails recorded at −60 mV after brief 30 msec step to −40mV from a holding potential of −90 mV (voltage record omitted for clarity). C, I–Vrelationships for I_Ba recorded in the absence and presence of 1 μm nociceptin.Asterisk marks characteristic shoulder onI–V relationship that results fromI_Ba,T. Note attenuation of current at −40 mV (I_Ba,T) and −10 mV (attenuation of HVA I_Ba) by nociceptin.D, I–V relationships forI_Ba recorded in the absence and presence of 1 μm morphine. Note lack of attenuation of current at potentials more negative than −40 mV (I_Ba,T, marked byasterisk) and attenuation of HVAI_Ba recorded at potentials positive to −20 mV. E, Log-concentration-response relationship for nociceptin. Numbers on graph indicate numbers of cells tested with each concentration; error bars, which indicate SEM, are smaller than data markers in some cases. The concentration of nociceptin required for half-maximum suppression of I_Ba,T(EC₅₀) is 100 nm. F, Data from E replotted as a Hill plot [Log₁₀R/R −R_m vs Log₁₀ nociceptin concentration ÷ EC₅₀; where Ris amplitude of response and R_m is the maximum response that can be obtained;K_D, the dissociation equilibrium constant for nociceptin and its receptor is assumed to equal the EC₅₀ (100 nm)]. Linear fit to data points yields a line with a gradient of 1 (Hill coefficient of 1.0).

Fig. 2.

Effects of nociceptin and morphine on HVAg_Ba. A₁,B₁, Currents recorded in response to a step from −90 to −10 mV in the presence and absence of 1 μmnociceptin or 1 μm morphine. Note suppression of current and slowed rate of activation in the presence of agonists.A₂, B₂, Removal of suppression of HVA g_Ba induced by 1 μm nociceptin or 1 μm morphine with depolarizing prepulses to +100 mV. Current responses to depolarizing commands are off scale; 2 nA/20 msec calibration inA₁ refers also to records inB₁, and 2 nA/40 msec calibration inA₂ refers also to records inB₂. Records in A₁and A₂ from the same cell and records inB₁ and B₂ from another cell.

Fig. 3.

Pharmacology of nociceptin attenuation of T-current. Superimposed data records show (A₁) suppression ofI_Ba,T by 1 μm nociceptin, (A₂) a second response to nociceptin recorded in the same cell in the presence of 1 μmnaloxone, (B₁) suppression ofI_Ba,T in another cell by 1 μmnociceptin, and (B₂) attenuation of nociceptin-induced inhibition in the second cell by 1 μmnalbzoh. I_Ba,T evoked by a step to −40 mV from holding potential of −90 mV (voltage record omitted for clarity).

Fig. 4.

Evidence against G-protein involvement in the effect of nociceptin on T-currents.A₁, Superimposed data records to show suppression of I_Ba,T by 100 nmnociceptin. A₂, Superimposed records from A₁ normalized and replotted. Note that the two records superimpose exactly, and the presence of nociceptin does not slow the onset or the rate of inactivation of the current.B₁, Graph of time course of an experiment done with a pipette containing 100 μmGTP-γ-S. Points represent amplitudes ofI_Ba,T or HVA I_Baevoked by a double-pulse protocol applied once every 20 sec. BothI_Ba,T and HVA I_Baare suppressed by 1 μm nociceptin. AlthoughI_Ba,T returns to control amplitude after nociceptin washout, HVA I_Ba remains suppressed throughout the course of the experiment. The cell had been left to stabilize with intracellularly applied GTP-γ-S for 15 min before the application of nociceptin.B₂, Original superimposed data record collected before, during, and after application of nociceptin in the experiment plotted in B₁. The cell was stepped from −90 to −40 mV to activateg_Ba,T and then to −10 mV to activate HVAg_Ba. Nociceptin invokes reversible suppression of the former and irreversible suppression of the latter.

Fig. 5.

Further evidence against G-protein involvement in the effect of nociceptin on T-currents. A, Effect of 1 μm nociceptin on I_Ba,T and HVAI_Ba in the same cell studied with 2 mm GDP-β-S in the pipette. The cell had been allowed to equilibrate with intracellularly applied GDP-β-S for 15 min before the application of nociceptin. Superimposed records acquired before and during nociceptin application show thatI_Ba,T was suppressed when HVAI_Ba was unaffected. B, An experiment similar to that of A but using AlF₃ (100 μm) instead of GDP-β-S in the pipette. The cell had been allowed to equilibrate with intracellularly applied AlF₃ for 20 min before the application of 1 μm nociceptin. Superimposed records acquired before, during, and after nociceptin application show thatI_Ba,T was reversibly suppressed when HVAI_Ba was unaffected. Voltage protocol inB applies also to experiment illustrated inA.

Fig. 7.

Effects of nociceptin, morphine, and Ni²⁺ on APs, bursting activity, and afterdepolarizations. All records were obtained from the same cell. APs were evoked from a 5 msec/0.3 nA depolarizing current command applied via the recording electrode. Superimposed records of bursts of APs were recorded before and during superfusion of (A) 1 μm nociceptin, (B) 1 μm morphine, and (C) 100 μm Ni²⁺. Note that afterdepolarization and burst of spikes are attenuated by nociceptin and Ni²⁺ but not by morphine. Calibration (40 mV/40 msec) in B and current trace in C apply to all records.