Tricyclic antidepressants dose-dependently modulate the biphasic activity of the TRPC5 channel through opioid receptors

Abstract

Tricyclic antidepressants (TCAs) have been widely used for the treatment of major depressive disorder and other psychiatric conditions. However, their clinical application has declined due to adverse side effects and the availability of newer antidepressants with improved safety profiles. TCAs primarily target serotonin and norepinephrine receptors but also interact with a variety of other receptors and ion channels, contributing to both their therapeutic and adverse effects. We previously reported that TCAs regulate transient receptor potential canonical type 4 (TRPC4) channels. In this study, we investigated whether TCAs similarly modulate TRPC5 channels. Using HEK293 cells overexpressing TRPC5, we measured TRPC5 currents and intracellular calcium levels. Without altering TRPC5 expression levels, TCAs (amitriptyline, desipramine, and imipramine) dose-dependently reduced inward currents through TRPC5, with IC₅₀ values of 2.9, 10.3, and 11.7 μM, respectively. Given that TCAs can act as off-target agonists at opioid receptors (ORs), we co-expressed TRPC5 with various OR subtypes (μ-, δ-, and κ-ORs). Our results revealed that at low concentrations, TCAs enhanced TRPC5 activation through OR stimulation, whereas at higher concentrations, competitive inhibition of TRPC5 activity predominated. The biphasic modulation of TRPC5 by TCAs may contribute to a wide spectrum of cardiovascular and neurological manifestations, depending on the dosage and clinical application. Overall, these findings enhance the pharmacological understanding of the molecular mechanisms underlying the actions of TCAs and emphasize the need for more targeted therapeutic approaches.

Keywords: Biphasic; Depression; Opioid receptor; TRPC5; Tricyclic antidepressant.

Fig. 1. Tricyclic antidepressants (TCAs) inhibit the activity of the TRPC5 channel.

All experiments were performed with coexpressed TRPC5 and μ-OR in HEK293 cells. Representative current trace (left), I-V curve (middle), and current amplitude (right) of TRPC5 treated with 10 μM TCAs. In representative current trace, the applications of antidepressants were above the traces as bars. (A) AMI (n = 7). (B) DES (n = 17). (C) IMI (n = 5). **p < 0.01, ***p < 0.001. TRPC5, transient receptor potential canonical type 5; OR, opioid receptor; I-V, current-voltage; AMI, amitriptyline; DES, desipramine; IMI, imipramine.

Fig. 2. Tricyclic antidepressants (TCAs) dose-dependently reduce the TRPC5 current without affecting channel expression.

(A-C) Concentration-inhibition curves for antidepressants obtained from TRPC5 currents. Hill coefficients were calculated from a Boltzmann sigmoid curve to averaged recordings as shown in curve. (A) IC₅₀ of 2.88 μM for AMI. (B) IC₅₀ of 10.31 μM for DES. (C) IC₅₀ of 11.68 μM for IMI. (D) Representative Western blot (upper) and the quantified data of the ratio (bottom) on surface expression level of TRPC5s (n = 5). Values are presented as mean ± SEM. TRPC5, transient receptor potential canonical type 5; AMI, amitriptyline; DES, desipramine; IMI, imipramine; NKA, sodium-potassium ATPase.

Fig. 3. Tricyclic antidepressants (TCAs) stimulate the opioid receptors, activating the TRPC5 channel.

(A, C, E) Representative current trace (left), I–V curve (middle), and current amplitude (right) of TRPC5 with coexpressed μ-OR treated with 10 μM TCAs. (A) AMI (n = 5). (C) DES (n = 8). (E) IMI (n = 5). (B, D, F) The current amplitude of TRPC5 with coexpressed δ- and κ-ORs treated with 10 μM TCAs. (B) δ-OR (n = 7) and κ-OR (n = 5). (D) δ-OR (n = 5) and κ-OR (n = 5). (F) δ-OR (n = 5) and κ-OR (n = 4). Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. TRPC5, transient receptor potential canonical type 5; OR, opioid receptor; I–V, current-voltage; AMI, amitriptyline; DES, desipramine; IMI, imipramine.

Fig. 4. Tricyclic antidepressants (TCAs) bind to the opioid receptors, partially activate Gi, and subsequently facilitate the TRPC5 current.

(A) Fluo-4 fluorescence trace (left), and peak F/F₀ (right) of intracellular [Ca²⁺]_i in TRPC5 overexpression in HEK293. (B) The peak F/F₀ of TRPC5 with co-expressed δ- and κ-ORs treated with 10 μM DES. (C-E) The expression of OR WT and the D-R mutant with the application of AMI (black) and agonist (gray). (C) DES; WT (n = 5) and D147R (n = 5), DAMGO; WT (n = 5) and D147R (n = 4). (D) DES; WT (n = 5) and D128R (n = 5), DPDPE; WT (n = 4) and D128R (n = 4). (E) DES; WT (n = 5) and D138R (n = 4), U50488; WT (n = 5) and D138R (n = 4). (F) Dose-dependent effects of DES on TRPC5 current amplitude with Gaussian fitting. (G) The current amplitude of TRPC5 with coexpressed μ-OR by pretreated 10 μM PTX for 16 h. (H) The current amplitude of TRPC5 with coexpressed μ-OR and Gα_i2(G203T). Values are presented as mean ± SEM. *p < 0.05, ***p < 0.001. TRPC5, transient receptor potential canonical type 5; OR, opioid receptor; AMI, amitriptyline; DES, desipramine; IMI, imipramine; WT, wild-type; PTX, pertussis toxin; n.s.; not significant.