Sigma-1 Receptor Plays a Negative Modulation on N-type Calcium Channel

Abstract

The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca²⁺ channel is still unclear. Considering both sigma-1 receptors and N-type Ca²⁺ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs) in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) and immunofluorescence staining. N-type Ca²⁺ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084) were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063). Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca²⁺ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca²⁺ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET) assays and co-immunoprecipitation (Co-IP) demonstrated that sigma-1 receptors and N-type Ca²⁺ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T) cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca²⁺ channels. The mechanism for the inhibition of sigma-1 receptors on N-type Ca²⁺ channels probably involved a chaperone-mediated direct interaction and agonist-induced conformational changes in the receptor-channel complexes on the cell surface.

Keywords: N-type Ca2+ channel; electrophysiology; ion channels modulation; protein–protein interaction; sigma-1 receptor.

FIGURE 1

Identification of the expression of N-type Ca²⁺ channel and sigma-1 receptor in ChIs. (A) An IR-DIC image of a striatal slice illustrating the characteristic appearance of giant cholinergic interneurons (ChIs). (B) Representative photograph of single-cell reverse transcription polymerase chain reaction (scRT-PCR). Experiment products were stained in a 2% agarose gel. Molecular mass markers were shown in the first lane. β-actin and GAPDH were introduced as positive control. The presence of ChAT indicated the aspirated cell was a ChI. (C) Bar plot indicated the co-expression of ChAT, sigma-1 receptor and α1A-C in ChIs by scRT-PCR. ChAT and sigma-1 receptor were in every detected neuron. The α1B subunit of N-type Ca²⁺ channel was found in 92.1% detected cells. (D) Immunofluorescence of successive paraffin-embedded rat striatum slice observed under 40× objective lens. Left: using the anti-sigma-1 receptor antibody at a dilution of 1:20 and FITC-conjugated affinipure goat anti-rabbit IgG. Middle: Using the anti-ChAT antibody at a dilution of 1:200 and TRITC-conjugated affinipure rabbit anti-goat IgG. Right: the merged picture of the sigma-1 receptor and ChAT.

FIGURE 2

Sigma-1 receptor agonist reduced currents of Ca²⁺ channel in ChIs. (A) Representative trace of Ca²⁺ channel current obtained from the rat striatum ChIs in the brain slice. (B) I–V plot of the recorded Ca²⁺ channel current trace in (A). (C–E) Representative traces of current evoked before and after the bath application of 0.5 μM CTX, 50 μM SKF-10047, and 50 μM Pre-084, respectively. (F) Summarized bar graph to present the effect of CTX (N-type Ca²⁺ channel blocker), different concentrations of SKF-10047 and Pre-084 (sigma-1 receptor agonists) on Ca²⁺ currents. (G) Summarized bar graph to present the effect of BD-1063 (sigma-1 receptor antagonist) on the Ca²⁺ current inhibition induced by SKF-10047. ^∗p < 0.05.

FIGURE 3

Expression of N-type Ca²⁺ channel and sigma-1 receptor in xenopus oocytes. (A) Upper panel: the current of N-type Ca²⁺ channel recorded in oocytes after microinjection with N-type Ca²⁺ channel cRNA. Lower panel: the current was blocked by N-type Ca²⁺ channel blocker CTX (0.2 μM). (B) I–V plot of the recorded N-type Ca²⁺ channel current in (A). (C) Schematic Western blot strap of oocytes injected with sigma-1 receptor cRNA (the first lane) and control group (the middle three lanes), which indicated the sigma-1 receptor expressed successfully. The last lane showed the Marker.

FIGURE 4

Currents recorded at different cRNA ratio of sigma-1 receptor to N-type Ca²⁺ channel. (A) Currents recorded from the oocytes injected with different cRNA ratio. The cRNA concentration ratio of Sigma-1 receptor: N-type Ca²⁺ channel = 1:1, 0.5:1, 0.25:1, 0.125:1, 0.0625:1, and 0.03125:1, respectively. (B) Summarized bar graph to present the N-type Ca²⁺ currents decreased with the increase of sigma-1 receptor cRNA injected in oocytes. The current amplitude recorded from oocytes with injection of N-type Ca²⁺ channel cRNA only was normalized as 100%. Values are Mean ± SEM (n = 18 for 1:1, n = 18 for 0.5:1, n = 17 for 0.25:1, n = 26 for 0.125:1, n = 17 for 0.0625:1, and n = 10 for 0.03125:1 group, respectively. ^∗∗∗p < 0.001, ^∗p < 0.05). (C) The expression levels of the two proteins from oocytes injected with the cRNA ratio of sigma-1 receptor: N-type Ca²⁺ channel at 0.03125:1, 0.0625:1, 0.125:1, 0.25:1, 0.5:1, and 1:1 from left to right. The blots in the right two lanes showed the protein expression in oocytes of control groups, which were injected with the N-type Ca²⁺ channel cRNA or the sigma-1 receptor cRNA alone.

FIGURE 5

Effect of sigma-1 receptor agonist and antagonist on N-type Ca²⁺ channel. (A) Example trace of the inhibition on N-type Ca²⁺ current induced by 50 μM SKF-10047. The oocyte was injected with a cRNA ratio of sigma 1R: N-type Ca²⁺ channel = 0.25:1. (B) I–V plot of the recorded N-type Ca²⁺ current from the oocyte in (A). (C) Summarized bar graph to present the effect of SFK-10047 (sigma-1 receptor agonist) and BD-1063 (sigma-1 receptor antagonist) on N-type Ca²⁺ currents. Two-way ANOVA analysis, Mean ± SEM, ^∗p < 0.05.

FIGURE 6

Exogenous expression of Sigma-1-Dsred receptor and Ca_v2.2-GFP in HEK-293T cell line. (A) Fluorescence images of HEK-293T cells transfected with 0.5 μg Sigma-1-Dsred receptor cDNA. (B) Fluorescence images of HEK-293T cells transfected with 0.5 μg Ca_v2.2-GFP (α1b+β1b+α2δ1subunits) cDNA. (C) Confocal microscopy images of HEK-293T cells with both 0.5 μg Ca_v2.2-GFP and 0.5 μg Sigma-1-Dsred receptor cDNA. Co-localization was shown in yellow.

FIGURE 7

Protein–protein interaction between sigma-1 receptors and N-type Ca²⁺ channels. (A) HEK-293T cells were transfected with both Ca_v2.2-GFP (α1b+β1b+α2δ1) and sigma-1-Dsred receptor. Pseudo-color images were obtained under the three filter sets: GFP (a), Dsred (b), and FRET (c). After subtraction of background and bleed-through signals, net FRET (d) was acquired. Normalized FRET (e) values were got by using equation described in section “Materials and Methods.” Color bars represented relative degree of net FRET and normalized FRET within the cells. (B) Representative cell enlarged from (e). The net FRET value of the point with the arrow was 0.81 ± 0.06 (Mean ± SD). (C) Bar graph of the statistical numbers in 11 experimental fields (203) and 5 negative control fields (0). (D) Co-immunoprecipitation of GFP-Ca_v2.2 channels and sigma-1-Dsred receptors. Total lysates were prepared from HEK-293T cells. Immunoprecipitated samples were run on the gels and the blots probed with either anti-Ca_v2.2 antibody or anti-sigma-1 receptor antibody.