T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids

Abstract

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC(50) approximately 200 nm for N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Figure 1.

N-Arachidonoyl amino acids inhibit Cav3 currents. A, Whole-cell calcium currents recorded from a tsA-201 cell expressing Cav3.2 channels in the absence (control, Ctrl), in the presence of 3 μm NAGly and after washout with a solution containing 3 mg/ml BSA. Currents were elicited by a depolarization at −30 mV (200 ms duration) applied every 5 s from −75 mV. B, Time course of the decrease in Cav3.2 current amplitude during NAGly application and subsequent washout with the BSA solution. C, Schematic structures of N-arachidonoyl amino acids containing either a serine (NASer), an alanine (NAAla), a γ-aminobutyric acid (NAGABA), or a 3-OH-γ-aminobutyric acid (NAGABA-OH) group as well as anandamide (NAEA) and N-arachidonoyl taurine (NATau). D, Summary of the effects of N-arachidonoyl amino acids and of NATau and NAEA (n = 14–27). E, F, Dose dependence of Cav3 current inhibition by NAGly (E, n = 5–20 per points) and NAGABA-OH (F, n = 3–18 per points). Cav3 current inhibition was fitted with the sigmoidal Hill equation.

Figure 2.

NAGABA-OH induces a strong shift in steady-state inactivation properties of Cav3.2 channels. A, Representative families of Cav3.2 currents recorded in the absence and presence of 1 μm NAGABA-OH. Effects of NAGABA-OH on current–voltage (I–V) curves of Cav3.2 currents is presented as an inset (n = 5–9). The current traces were elicited by a series of step depolarizations ranging from −80 to +10 mV (200 ms duration) from −80 mV. B, Similar inhibition of inward and outward Cav3.2 currents by NAGABA-OH (n = 5–11). In these experiments capacitive and background currents were subtracted by a −P/8 protocols. C, Cav3.2 current inhibition by NAGABA-OH is not frequency dependent (n = 13–14). Currents were elicited by a depolarization at −30 mV (200 ms duration) applied from −75 mV at a frequency of 1 or 0.1 Hz. D, Cav3.2 currents elicited at −30 mV from HPs ranged from −110 mV to −60 mV (10 s duration, 5 mV increments) in the absence and presence of 3 μm NAGABA-OH. E, Steady-state inactivation curves obtained from experiments illustrated in (D). Data were fitted with the Boltzmann equation (n = 10–15). F, No significant effect of NAGABA-OH when applied at a HP of −110 mV.

Figure 3.

Inhibition of Cav3.2 currents by NAGABA-OH in cell-free outside-out patches. A, Effects of 3 μm NAGABA-OH on Cav3.2 currents recorded in an outside-out patch. Currents were elicited by a voltage ramp of 100 ms duration from −90 mV to +40 mV applied every 5 s from −90 mV. B, Time course of the decrease in Cav3.2 current amplitude during NAGABA-OH application and subsequent washout with the BSA solution. C, Summary of the effects of NAGABA-OH on Cav3.2 currents obtained in outside-out patch configuration (n = 10–15).

Figure 4.

Effects of NAGly and NAGABA-OH on recombinant Cav1.2, Cav2.2, Nav1.7, Nav1.8, TASK1, and TRPV1 channels. A–C, Whole-cell currents recorded from tsA-201 cells expressing Cav1.2 channels (A), Cav2.2 (B), and Nav1.7 (C) in the absence and presence of 3 μm NAGly and 3 μm NAGABA-OH. Currents were elicited by a depolarization at 0 mV (200 ms duration for Cav1.2 and Cav2.2 or 20 ms for Nav1.7) applied every 5 s from −75 mV. D, Effects of 3 μm NAGly and 3 μm NAGABA-OH on whole-cell currents recorded from a F11 cell expressing Nav1.8. Currents were elicited in the presence of 0.5 μm TTX by a depolarization at 0 mV (30 ms) applied every 5 s from −65 mV. E, Inhibition of TASK1 currents by 3 μm NAEA but not by 3 μm NAGly and NAGABA-OH. TASK1 currents are inhibited by a mild extracellular acidification, pH 6.5. Currents were elicited by voltage ramps of 1 s duration from −100 to 0 mV applied every 5 s from −80 mV. F, G, Summary of the effects of 1–3 μm NAGly (F, n = 6–28) and NAGABA-OH (G, n = 6–19). H, Activation of TRPV1 by 10 μm NAEA but not by 10 μm NAGly and 10 μm NAGABA-OH. Intracellular calcium measurements were performed using Fluo4-AM and drugs were applied at the time indicated by an arrow. All measurements were performed on four independent 96-well plates and were normalized to the response obtained with 1 μm capsaicin (caps.) I, 10 μm ruthenium red (RR) but not 10 μm NAEA, NAGly, and NAGABA-OH inhibits TRPV1 activation by 1 μm capsaicin. Similar experiments as in H except that Ctrl indicates the capsaicin response obtained in untransfected cells (for clarity errors bars were omitted).

Figure 5.

Inhibition of Cav3.2 in sensory neurons underlies NAGly-induced analgesia. A, B, Calcium currents recorded (in the absence of sodium) from DRG neurons in the absence (Ctrl) and presence of 3 μm NAGly and after washout with a solution containing 3 mg/ml BSA. Currents were elicited by a depolarization (200 ms) at −30 mV (A, T-type) or at 0 mV (B, HVA) applied every 5 s from −75 mV. C, D, Effects of NAGly on sodium currents in DRG neurons. Sodium currents were recorded in the presence of 10 μm La³⁺ (a calcium channel blocker) with [D, I(Na) TTX-r] or without [C, I(Na) total] 0.5 μm TTX. Currents were elicited by a depolarization at 0 mV (20 ms duration) applied every 5 s from −70 mV (C) or −50 mV (D). E, Summary of the effects of NAGly on calcium and sodium currents in DRG neurons (n = 10–12). To limit the contamination of T-currents by HVA currents, and vice versa, HVA currents were measured as the remaining current after 150 ms, whereas T-currents were measured as the difference between the peak current and the remaining current after 150 ms. F, G, Effects of vehicle and NAGly solution on thermal pain (46°C) when injected in the hindpaw in WT (F) and Cav3.2 KO mice (G). Results are expressed as the PWL as a function of the time after injection. The “Pre” values represent the values obtained before injection. H, Summary of the data obtained in WT mice 10 min after injection of vehicle, BSA, NAGly, NAGABA-OH, and morphine (n = 8–10 mice per bars). Effect of NAGABA-OH on PWL in contralateral (contra.) is also reported in WT mice (n = 9). I, J, Effects of vehicle and NAGABA-OH solution on thermal pain (46°C) when injected in the hindpaw in WT (I) and Cav3.2 KO mice (J). Effect of NAGABA-OH on PWL in contralateral is also reported for WT mice as indicated (I). K, Summary of the data obtained in Cav3.2 KO mice 10 min after injection of vehicle, BSA, NAGly, NAGABA-OH, and morphine (n = 8–10 mice per bars).