Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels

Abstract

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.

Fig. 1.

Progesterone binding to σ-receptors. A: radioligand binding curves demonstrating the competitive displacement by progesterone of ligand binding to σ₁- and σ₂-receptors. (+)[³H]-pentazocine was used as a ligand for σ₁-receptors. [³H]ditolylguanidine ([³H]DTG) with cold (+)pentazocine (μM) was used to resolve σ₂-receptors. The binding affinity of progesterone was determined to be 239 nM for σ₁-receptors and 441 nM for σ₂-receptors. B: σ-Receptor photolabeling with the σ₁/σ₂-receptor photolabel 1-N-(2′,6′-dimethyl-morpholino)-3-(4-azido-3-[¹²⁵I]iodo-phenyl)propane ([¹²⁵I]IAF) indicated that σ₁- and σ₂-receptors are expressed in human embryonic kidney (HEK)293 cells. The first lane represents HEK293 cell homogenates alone [control (Cont)], the second in 10 μM (+)pentazocine (Pent), the third in 20 μM DTG, and the fourth in 50 μM progesterone (Prog).

Fig. 2.

Progesterone actions on Na⁺ channel inhibition by σ-receptor ligands in HEK293 cells. Na⁺ currents evoked by voltage steps from −80 mV to −10 mV before and after addition of 10 μM DTG, 10 μM PB28, and 100 μM (+)SKF10047. A and B: current inhibition by σ-receptor ligands in the absence of progesterone (A) and in the presence of 10 μM progesterone (B).

Fig. 3.

Progesterone actions on Na⁺ channel inhibition by σ-receptor ligands in HEK293 cells with σ₁-receptor small interfering RNA (siRNA) knockdown. A and B: Na⁺ currents evoked by voltage steps from −80 mV to −10 mV recorded from HEK293 cells transfected with sig1RsiRNA in the absence and presence of 10 μM DTG, 10 μM PB28, and 100 μM (+)SKF10047 before (A) and after (B) the addition of 10 μM progesterone.

Fig. 4.

Comparison of Na⁺ current inhibition with and without progesterone in control HEK293 cells and HEK293 cells with σ₁-receptor siRNA knockdown. Average inhibition of each ligand was determined from current traces such as shown in Figs. 2 and 3. C, control; P, 10 μM progesterone. Bars represent means ± SE for n = 4–7. The values for DTG and PB28 differ significantly in both control HEK293 cells and with the addition of sig1RsiRNA, between recordings with and without 10 μM progesterone (*P < 0.005).

Fig. 5.

Progesterone actions on Na⁺ current inhibition by N,N-dimethyltryptamine (DMT) in HEK293 cells. A and B: Na⁺ currents evoked by voltage steps from −80 mV to −10 mV recorded from control HEK293 cells (A) and cells with reduced σ₁-receptor expression (B) by transfection with sig1RsiRNA in the absence and presence of 100 μM DMT before and after the addition of 10 μM progesterone. C: average Na⁺ current inhibition by DMT in each condition. Bars represent means ± SE for n = 5–7. The percent inhibition differs significantly in control HEK293 cells between recordings with and without 10 μM progesterone (*P < 0.005).

Fig. 6.

Concentration dependence of σ-receptor antagonism by progesterone in HEK293 cells. Na⁺ current evoked by voltage steps from −80 mV to −10 mV, before and after addition of the indicated concentration of agonist, in the presence of various concentrations of progesterone. Peak current was normalized to control and plotted versus drug concentration. The horizontal lines indicate Na⁺ current inhibition by agonist in the absence of progesterone. Data points represent means ± SE for n = 5–7 cells. Curves represent best fits to the data using the single-site saturation equation given in methods. Half-maximal inhibition (IC₅₀) values were 105, 380, 620, and 0.3 nM for DTG, PB28, DMT, and (+)SKF10047, respectively.