σ-1 receptor at the mitochondrial-associated endoplasmic reticulum membrane is responsible for mitochondrial metabolic regulation

Article abstract of DOI:10.1124/jpet.112.198168

The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a small section of the outer mitochondrial membrane tethered to the ER by lipid and protein filaments. One such MAM protein is the σ-1 receptor, which contributes to multiple signaling pathways. We found that short interfering RNA-mediated knockdown of σ-1 reduced pregnenolone synthesis by 95% without affecting expression of the inner mitochondrial membrane resident enzyme, 3-β-hydroxysteroid dehydrogenase 2. To explore the underlying mechanism of this effect, we generated a series of σ-receptor ligands: 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2- carboxamide (KSCM-1), 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran- 2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide (KSCM-11) specifically bound to σ-1 in the nanomolar range, whereas KSCM-5 and KSCM-11 also bound to σ-2. Treatment of cells with the KSCM ligands led to decreased cell viability, with KSCM-5 having the most potent effect followed by KSCM-11. KSCM-1 increased σ-1 expression by 4-fold and progesterone synthesis, whereas the other compounds decreased progesterone synthesis. These differences probably are caused by ligand molecular structure. For example, KSCM-1 has two methoxy substituents at C-5 and C-6 of the benzofuran ring, whereas KSCM-11 has one at C-6. KSCM ligands or σ-1 knockdown did not alter the expression of ER resident enzymes that synthesize steroids. However, coimmunoprecipitation of the σ-1 receptor pulled down voltage-dependent anion channel 2 (VDAC2), whose expression was enhanced by KSCM-1. VDAC2 plays a key role in cholesterol transport into the mitochondria, suggesting that the σ-1 receptor at the MAM coordinates with steroidogenic acute regulatory protein for cholesterol trafficking into the mitochondria for metabolic regulation. Copyright

Full text of DOI:10.1124/jpet.112.198168

1521-0103/12/3433-578–586$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
JPET 343:578–586, 2012
Vol. 343, No. 3
198168/3803519
␴-1 Receptor at the Mitochondrial-Associated Endoplasmic
Reticulum Membrane Is Responsible for Mitochondrial
□
S
Metabolic Regulation
Karla-Sue C. Marriott, Manoj Prasad, Veena Thapliyal, and Himangshu S. Bose
Department of Natural Sciences, College of Science and Technology, Savannah State University, Savannah, Georgia
(K.-S.C.M.); and Departments of Biochemistry and Biomedical Sciences, Mercer University School of Medicine and Memorial
University Medical Center, Anderson Cancer Institute, Savannah, Georgia (M.P., V.T., H.S.B.)
Received July 6, 2012; accepted August 23, 2012
ABSTRACT
The mitochondria-associated endoplasmic reticulum (ER) whereas KSCM-5 and KSCM-11 also bound to ␴-2. Treatment of
membrane (MAM) is a small section of the outer mitochon- cells with the KSCM ligands led to decreased cell viability, with
drial membrane tethered to the ER by lipid and protein KSCM-5 having the most potent effect followed by KSCM-11.
filaments. One such MAM protein is the ␴-1 receptor, which KSCM-1 increased ␴-1 expression by 4-fold and progesterone syn-
contributes to multiple signaling pathways. We found that thesis, whereas the other compounds decreased progesterone syn-
short interfering RNA-mediated knockdown of ␴-1 reduced thesis. These differences probably are caused by ligand molecular
pregnenolone synthesis by 95% without affecting expression structure. For example, KSCM-1 has two methoxy substituents at
of the inner mitochondrial membrane resident enzyme, 3-␤- C-5 and C-6 of the benzofuran ring, whereas KSCM-11 has one at
hydroxysteroid dehydrogenase 2. To explore the underlying C-6. KSCM ligands or ␴-1 knockdown did not alter the expression of
mechanism of this effect, we generated a series of ␴-recep- ER resident enzymes that synthesize steroids. However, coimmuno-
tor ligands: 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperi- precipitation of the ␴-1 receptor pulled down voltage-dependent
din-1-yl)propyl)benzofuran-2-carboxamide (KSCM-1), anion channel 2 (VDAC2), whose expression was enhanced by
3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran- KSCM-1. VDAC2 plays a key role in cholesterol transport into the
2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N- mitochondria, suggesting that the ␴-1 receptor at the MAM coordi-
phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide nates with steroidogenic acute regulatory protein for cholesterol traf-
(KSCM-11) specifically bound to ␴-1 in the nanomolar range, ficking into the mitochondria for metabolic regulation.
ates with the endoplasmic reticulum (ER), thereby promoting
Introduction
the rapid reuptake of extracellular Ca2ϩ to replenish ER
The physical interaction that occurs between subcellular
stores (Blaustein and Golovina, 2001; Va´rnai et al., 2007).
organelles provides an efficient mechanism for modulating
The mitochondria, another major Ca2ϩ-buffering organelle,
cellular processes. For example, plasma membrane associ-
also associates with the plasma membrane, and by doing so,
interacts with the ER (Malli et al., 2003). The physical inter-
H.S.B. was supported by the National Institutes of Health [Grant
action between the ER and mitochondria forms a structural
element referred to as the mitochondria-associated ER mem-
brane (MAM) (Vance, 1990), which plays a significant role in
cellular functions, including lipids and cholesterol transport,
energy metabolism, and apoptosis (Miller and Bose, 2011).
The MAM, present in many cell types, typically consists of
only a small section of the outer mitochondrial membrane
(OMM) that associates with the ER (Csorda´s et al., 2006).
HD057876] and the Anderson Cancer Institute. K.-S.C.M. was supported by
the National Institutes of Health National Institute on Drug Abuse [Grant
DA027086] and the National Institutes of Health National Center on Minority
Health and Health Disparities [Grant 5P20MD003941] (to K.-S.C.M. and
H.S.B.).
K.-S.C.M. and M.P. contributed equally to this work.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
http://dx.doi.org/10.1124/jpet.112.198168.
□S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: ER, endoplasmic reticulum; MAM, mitochondria-associated ER membrane; Ab, antibody; IMM, inner mitochondrial membrane;
IP3, inositol 1,4,5-triphosphate; KSCM-1, 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl) benzofuran-2-carboxamide; KSCM-5,
3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide; KSCM-11, 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)
benzofuran-2-carboxamide; OMM, outer mitochondrial membrane; SCC, side-chain cleavage; siRNA, short interfering RNA; SKF-10047, 2S-
[2␣,6␣,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol; StAR, steroidogenic acute regulatory protein;
TLC, thin-layer chromatography; VDAC, voltage-dependent anion channel; 3␤HSD2, 3-␤-hydroxysteroid dehydrogenase.
578

␴-1 Regulates Mitochondrial Enzymatic Activity
579
Initially, the literature reported a distance of approximately longed calcium signaling into the mitochondria via these IP3
100 nm between the ER and the OMM; however, a study receptors. There are possible explanations where the IP3
using electron tomography demonstrated that the distance is receptors are connected from the MAM through VDAC with
actually within 10 to 25 nm (Csorda´s et al., 2006). Given the the OMM, because of the short distance of 20 nm. Interaction
roles of MAM in cellular physiology, it is not surprising that of ␴-1 receptor is facilitated with the Ca2ϩ channel at the
this membrane interface is enriched with a variety of pro- mitochondrial membrane either directly or through the
teins, including enzymes associated with lipid and glucose VDAC (Hayashi and Su, 2007). It is noteworthy that proges-
metabolism, such as phosphotidylserine synthase, phospho- terone synthesis in the bovine placentome is calcium-depen-
tidylethanoloamine methyltransferase 2, acyl-CoA choles- dent (Shemesh et al., 1984). Together, this evidence points to
terol acyltransferase, diacylglycerol acyltransferase, and glu- a possible role for ␴-1 receptors in steroidogenesis.
cose 6-phosphate (Hayashi et al., 2009). Other proteins
Selective ligands for the ␴-1 receptor can help elucidate the
shown to localize to the interface include the chaperones physiological roles of this protein. ␴-1 Ligands must contain
calnexin and calreticulun, which both bind Ca2ϩ and help a basic amine and two hydrophobic appendages (Costantino
with the quality control of protein folding; phosphofurin et al., 2005). Hence, a common pharmocophoric feature of ␴-1
acidic cluster sorting protein 2, which plays a role in apopto- ligands is an N-alkyl, N,N-dialkyl, or N-arylalkyl amine moi-
sis, and the ␴-1 receptor, which has been linked to a multi- ety (Ablordeppey et al., 2000), which poses a challenge in the
tude of signaling pathways (Simmen et al., 2005; Hayashi synthesis of these ligands. It is noteworthy that progester-
and Su, 2007; Hayashi et al., 2009; Kornmann et al., 2009). one, which lacks a basic nitrogen, is considered one of the
␴-Receptors consist of two subtypes, ␴-1 and ␴-2; however, putative endogenous ligands for the ␴-1 receptor (Walker et
only ␴-1 has been characterized at the molecular level. Ini- al., 1990). N,N-dimethyltryptamine has been identified as a
tially classified as a type of opioid receptor, the ␴-1 receptor potential endogenous ␴-1 receptor ligand, but the role of
is now considered a nonopioid receptor (Su et al., 1988), but N,N-dimethyltryptamine as a ␴-1 receptor modulator is un-
a definitive identity of its endogenous ligand remains some- clear because of the low abundance at physiological concen-
what elusive. The ␴-1 receptor can be found in the central trations in brain tissues (Fontanilla et al., 2009). We synthe-
nervous system as well as in peripheral organs, including the sized ligands that have a benzofuran-2-carboxamide structural
lung, liver, adrenal gland, spleen, and pancreas (Hayashi and moiety, which has been N-arylated and N-alkylated to include
Su, 2007). Studies have shown that the ␴-1 receptor, which both N-phenyl and N-(3-(piperidin-1-yl) propyl substituents.
has two transmembrane-spanning regions (Jbilo et al., 1997; These ligands have a defined conformation and can be delivered
Aydar et al., 2002), translocates during the activation of to the ER in the cell. Using these ligands, we have identified an
multiple signaling pathways and modulates the function or interaction between the ␴-1 receptor and VDAC2 that contrib-
production of various intracellular secondary messengers utes to the regulation of mitochondrial pregnenolone synthesis.
(Hayashi and Su, 2001; Aydar et al., 2002), including binding to
neuroactive steroids, such as progesterone and dehydroepi-
androsterone (Valade et al., 2011). The ␴-1 receptor also helps
Materials and Methods
proteins fold appropriately at the ER to regulate the metabolic
Cell Culture, Isolation, and Purification of Mitochondria.
The mouse Leydig cell line (MA-10) was grown in Waymouth media
containing 15% horse serum, 5% fetal bovine serum, and 1ϫ genta-
mycin, which was replaced with 250 mg/ ml G418 (geneticin) for
VDAC1 knockdown (⌬VDAC1) MA10 cells (Bose et al., 2008). Cells
were maintained at 37°C in a humidified incubator under 5% CO2.
To isolate mitochondria, cells were removed from tissue culture
activity of receptor-mediated Ca2ϩ influx (Hayashi and Su,
2007).
Tissues generate steroid hormones on demand after trans-
port of cholesterol, which serves as the initial substrate, into
the mitochondria. Fractions of MAM that contain ␴-1 recep-
tors (Hayashi and Su, 2007) are exceptionally enriched in
cholesterol and neutral lipids (Hayashi and Su, 2003). More- plates by gentle scrapping in phosphate-buffered saline at room
temperature, incubated in hypotonic buffer (10 mM HEPES, pH 7.4)
for 30 to 40 min, and mitochondria were isolated following previously
described procedures (Bose et al., 2007; Pawlak et al., 2011b). The
mitochondrial preparation was verified by Western blotting with the
cytochrome P450 SCC enzyme (Corgen, Tapai, Taiwan) and calnexin
antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Ligand Concentration and Cell Viability Assay. The ␴-recep-
tor compounds act as ligands and are named “ligands” in this article.
Stock solutions (1 mM) of the ligands were made in 1ϫ phosphate-
buffered saline, filter-sterilized, and then further diluted up to 1 ␮M
over, acyl-CoA cholesterol acyltransferase, the enzyme re-
sponsible for the synthesis of cholesteryl esters, also resides
in the MAM. Thus, MAM might serve as a site for cholesterol
loading through the OMM. The fact that cholesterol trans-
port into the mitochondria represents the rate-limiting step
in steroidogenesis supports the potential importance of
MAM. Cholesterol is stored in lipid droplets as cholesterol
esters and is transported first from the OMM to the inner
mitochondrial membrane (IMM), where it is catalyzed by the
cytochrome P450 side-chain cleavage (SCC) enzyme, to form with serum-free medium. Cell viability was determined by the 3-[4,5-
dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide assay using a
commercially available kit following the manufacturer’s instructions
(Promega, Madison, WI). MA-10 cells were plated in a 96-well plate
at an initial density of 4 ϫ 103 cells per well for 24 h at 37°C in 5%
CO2 atm. After 24 h of serum starvation, the culture media were
changed to serum-free media containing 1, 5, 10, 50, and 100 nM
concentration for each KSCM ligand. Next, the cells were incubated
for 2 to 3 h with 0.5 mg/ml of 3-[4,5-dimethylthiazol-2yl]-2,5-diphe-
nyltetrazolium bromide reagent and lysed with dimethyl sulfoxide,
and the absorbance was measured at 550 nm (FlexStation 3; Molec-
pregnenolone, the first steroid in the biosynthetic pathway.
Pregnenolone is then transported back to the ER for the
synthesis of several other steroids (Miller and Bose, 2011).
Transport of cholesterol to the site of steroidogenesis is facil-
itated by steroidogenic acute regulatory protein (StAR),
which interacts with voltage-dependent anion channel 1
(VDAC1) at the OMM (Bose et al., 2008). VDAC may also
contribute to the association of inositol 1,4,5-triphosphate
(IP3) receptors with the OMM at the MAM. During times of
calcium depletion in the ER, the ␴-1 receptor induces pro- ular Devices, Sunnyvale, CA).

580
Marriott et al.
Western Blot Analysis. Protein (12.5 ␮g) was separated by 15% carboxamides (Scheme 1) with sodium hydride producing KSCM-11,
SDS-polyacrylamide gel electrophoresis and transferred to a polyvi- KSCM-1, and KSCM-5, respectively.
The structures of the new ligands were confirmed by 1H and 13C
nylidine difluoride membrane (Millipore Corporation, Billerica, MA).
The membrane was blocked with 3% nonfat dry milk for 45 min,
probed overnight with the primary antibodies, and then incubated
with peroxide-conjugated goat anti-rabbit or anti mouse IgG
(Thermo Fisher Scientific, Waltham, MA). Signals were developed
with chemiluminescent reagent (Thermo Fisher Scientific). For load-
ing control of all Western blots we stained the membrane with
␤-actin antibody, which was previously independently stained with
StAR or 3-␤-hydroxysteroid dehydrogenase (3␤HSD2) or VDAC1 and
VDAC2 antiserum. The band intensity was determined by using
Image Quant 5.2 (Phosphorimager; GE Healthcare, Chalfont St.
Giles, Buckinghamshire, UK). The densitometric calculation in each
figure reflects the ratio of ␴-1 or VDAC2 and ␤-actin following an
established procedure (Pitre et al., 2007).
Metabolic Conversion Assays and Analysis of Steroids. To
measure the conversion of [3H]pregnenolone to [3H]progesterone,
isolated mitochondria from steroidogenic MA-10 cells (300 ␮g)
were incubated in phosphate buffer along with the substrate, and
the reaction was initiated by the NAD (Pawlak et al., 2011a). For
metabolic conversion of [14C]cholesterol to pregnenolone, the re-
action was initiated by the addition of NADPH (Pawlak et al.,
2011a), and for complete conversion we used 5-fold excess of cold
carrier to reach the saturation point. The steroids were extracted
with ether/acetone (9:1 v/v), and equal amounts of a cold preg-
nenolone-progesterone (50:50; Sigma, St. Louis, MO) mixture in
CH2Cl2 was added as a carrier. The extracts were concentrated
under a stream of nitrogen or air and then separated by thin-layer
chromatography (TLC) (Whatman, Clifton, NJ) using a chloro-
form/ethyl acetate (3:1) mobile phase. The spots extracted from
TLC plates were subjected to gas chromatography-mass spectrom-
etry analysis on an Agilent 7890 GC with 5975C mass spectrom-
eter (Agilent Technologies, Santa Clara, CA) and analyzed as
described previously (Prasad et al., 2012).
␴-Receptor Ligand Synthesis. Our novel carboxamide ligands
[5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl) benzo-
furan-2-carboxamide (KSCM-1), 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)
propyl)benzofuran-2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N-
phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide (KSCM-
11)] maintain the characteristic pharmacophore of documented ␴-1 recep-
tor ligands, such as a basic alkylamine moiety, the N-(3-(piperidin-1-yl)
propyl with a protonatable nitrogen, and an aromatic phenyl ring and
aromatic benzofuran moiety as the two hydrophobic residues. Syntheses of
KSCM-11 and KSCM-1 and the unsubstituted KSCM-5 were achieved by
treating the corresponding carboxamides (Scheme 1) with NaH followed by
N-alkylation with iodopropylpiperidine outlined in Scheme 1. A halogen
exchange reaction was used to convert chloropropylpiperidine hydrogen
chloride salt in the presence of potassium iodide, tetrabutylammonium
bromide, and potassium carbonate into the more reactive iodopropylpip-
eridine in situ, which then reacted with anions obtained from treating
NMR spectra on a JEOL (Tokyo, Japan) 300-MHz spectrometer, and
high-resolution mass spectrometry data were obtained in the Mass
Spectrometry Laboratory, School of Chemical Sciences, University of
Illinois (Urbana IL). Chemical shifts for 1H and 13C NMR spectra are
reported in ␦ values (ppm) relative to an internal standard of tetram-
ethylsilane in CDCl3. Multiplicities are presented as follows: s ϭ
singlet, d ϭ doublet, t ϭ triplet, q ϭ quartet, qt ϭ quintuplet, and
m ϭ multiplet. Solvents were purified by using standard procedures.
TLC analyses were performed on Fluka (Buchs, Switzerland)
200-␮m silica gel, particle size 25 ␮m, F254 plates and visualized by
quenching of UV fluorescence (␭max ϭ 254 nm). All new ligands were
purified on a Biotage (Charlotte, NC) Isolera-4 flash purification
system using the indicated SNAP cartridges and solvents as eluents.
Radioligand Binding Assay. The primary binding screening
assays were performed by using concentrations ranging from 50 ␮M
(initial) to 10 ␮M (final) concentration of reference and experimental
ligands following standard procedure (Kova´cs and Larson, 1998), at
the National Institute of Mental Health-Psychoactive Drug Screen-
ing Program (http://pdsp.med.unc.edu/pdspw/function.php). The per-
centage of inhibition of specific binding by the experimental ligand
was determined. Secondary binding assays were used to determine
the inhibitor constant (Ki) of each of the ligands for ␴-1 and ␴-2
receptors. Ki represents the concentration of the competitor that
binds to half the enzyme sites at equilibrium in the absence of
substrate or other competitors (Kakkar et al., 2000). The log IC50
(the log of the ligand concentration that reduces radioligand binding
by 50%) was thus estimated from the data and used to obtain the Ki
by applying the Cheng-Prusoff approximation (Cheng and Prusoff,
1973).
␴-1 Receptor Knockdown and Coimmunoprecipitations.
siRNA oligonucleotides for ␴-1 were obtained from Ambion (Austin,
TX). COS-1 and MA-10 cells were transfected with 30 and 60 pmol of
siRNA1 (5Ј-GGACAUACAUACUUGUACAtt- 3Ј); siRNA2 (5Ј-
GGAGAGACAGUUGUACACGtt-3Ј); siRNA3 (5Ј-CGAUACUGGGCU-
GAGAUUUtt-3Ј), and siRNA4 (5Ј-GCUUACCACCUACCUCUUUtt-
3Ј), using oligofectamine (Invitrogen, Carlsbad, CA). Nontargeting
siRNA served as a control in all experiments. For coimmunoprecipita-
tion experiments, specific antibodies were preincubated with protein A
Sepharose CL-4B (0.5 ␮g/␮l; GE Healthcare) in 100 ␮l of 1ϫ coimmu-
noprecipitation buffer (1% Triton X-100, 200 mM NaCl, and 0.5% so-
dium deoxycholate) for 2 h at 4°C, and then beads were washed with
buffer. The antibody-coated beads were processed for coimmunoprecipi-
tation as described previously (Pawlak et al., 2011b), and immune
complexes were analyzed by Western blotting.
Results
Effect of ␴-1 Receptor Knockdown on Mitochondrial
and ER Protein Expression. To evaluate the potential role
of ␴-1 receptor in steroid synthesis (Fig. 1A), we first
generated four siRNAs (siRNA 1–4) targeting ␴-1 receptor
expression and tested their effects at 30 and 60 pmol in
MA-10 cells. Although all four reduced expression of the
␴-1 receptor at 60 pmol, only siRNA4 had an effect at 30
pmol (Fig. 1B), thus we continued our experiments using
siRNA4. We next asked whether loss of the ␴-1 receptor
would modulate the expression of other proteins associated
with the ER and mitochondria, such as ␤-actin (Fig. 1B,
right), 3␤HSD2 (Pawlak et al., 2011b; Prasad et al., 2012),
and OMM-associated Tom20 (Bose et al., 2002). Figure 1B,
right shows the indicated corresponding control experi-
ment for Fig. 1B, left. The results (Fig. 1B) showed that ␴-1
R2
R1
R2
R1
N
N
H
O
O
O
O
N
1a, R1 = OCH3 R2 = H
1b, R1 = OCH3 R2 = OCH3
1c, R1 = H
KSCM-11 R1 = OCH3 R2 = H
KSCM-1 R1 = OCH3 R2 = OCH3
KSCM-5 R1 = H
R2 = H
R2 = H
Reagents: NaH, 1-(3-chloropropyl)piperidine.HCl, K2CO3, KI, TBAB, CH2Cl2
Scheme 1. Synthesis of different KSCM ligands from carboxamides via
N-alkylation. The substitution of side chains is indicated above, generat-
ing KSCM-1, KSCM 5, and KSCM-11 ligands.

␴-1 Regulates Mitochondrial Enzymatic Activity
581
A
B
30 60 30 60 30 60 30 60
30 60 30 60 30 60 30 60
β-Actin Ab
β-Actin Ab
StAR
Sigma-1 Ab
42
42
29
42
3βHSD2 Ab
42
β-Actin Ab
20
Tom20 Ab
Sigma-1 Mitochondria Steroids
+NADPH
F
D
E
C
25
20
Chol
Preg
15
10
SCC Ab
Calnexin Ab
60
75
5
0
Cytosol
ER-Lumen
Origin
Mito + Trilo
-
+
+
+
+
+ NADPH
+ MA10 Mito + Trilo
Fig. 1. Expression and activity of the ␴-1 receptor in ␴-1 receptor-knockdown MA-10 cells. A, schematic presentation of a simplified version of the
biosynthesis pathway and its relationship to the MAM with ␴-1 receptor. B, left, all four siRNA oligonucleotides at 60 pmol reduced expression of the
␴-1 receptor in MA-10 cells, but only siRNA4 reduced expression at 30 pmol. Western blot shows unchanged expression of IMM resident 3␤HSD2 and
OMM-associated Tom20 in MA-10 cells. Right, loading control of probing the corresponding left with ␤-actin. C, isolated mitochondria from MA-10
control and siRNA knockdown cells were incubated with [14C]cholesterol and NADPH in a metabolic conversion assay, and metabolites were separated
by thin-layer chromatography. D, quantitative estimation of the metabolites from C. E, Western blot of the cellular fractions with the indicated
antibodies, showing the purity of our mitochondrial fraction. Data presented are the mean Ϯ S.E.M. of three independent experiments. F, proposed
model of interaction between ER and mitochondria through the MAM resident ␴-1 receptor.
receptor knockdown did not alter the expression of ER and gested that the ␴-1 protein plays a substantial role in steroid
mitochondrial resident proteins.
hormone synthesis.
Effect of ␴-1 Receptor Knockdown on Mitochondrial
Chemical Structure Elucidation of Ligands. We next
Pregnenolone Synthesis. Given the role of ␴-1 receptor as elucidated the chemical structure of the ligands by using
a chaperone at the ER-mitochondria interface to regulate NMR. The NMR of KSCM-1 showed 1H NMR (300 MHz,
Ca2ϩ signaling, and the requirement of Ca2ϩ for progester- CDCl3) ␦ 1.38 to 1.56 (m, 6H), 1.81 to 1.91 (qt, J ϭ 7.58 Hz,
one synthesis in the bovine placentome (Shemesh et al., 2H), 2.31 to 2.36 (m, 6H), 2.34 (s, 3H), 3.80 (s, 3H), 3.87 (s,
1984), we next wanted to explore the effect of ␴-1 knockdown 3H), 3.86 to 3.93 (m, 2H), 6.52 (s, 1H), 6.81 (s, 1H), 7.10- 7.30
on pregnenolone synthesis. We treated MA-10 cells with 30 (m, 5H). 13C NMR (300 MHz, CDCl3) ␦ 9.9, 10.1, 24.5, 25.3,
pmol of siRNA4 or siRNA4-isolated mitochondria, and then 26.0, 29.7, 49.0, 54.6, 56.2, 56.3, 56.6 94.7, 100.8, 120.8,
determined the conversion of cholesterol to pregnenolone in 122.4, 126.5, 126.9, 128.9, 143.2, 143.6, 146.7, 148.4, 149.9,
an in vitro metabolic conversion assay (Fig. 1, C and D). 161.4. The calculated and observed molecular mass [mass
Cytochrome P450 SCC enzyme (Chung et al., 1986) is an spectrometry (electrospray ionization)ϩ] for C26H33N2O4
inner mitochondrial resident protein, and calnexin is associ- [MϩH]ϩwas 437.2440.
1
ated with the ER. Thus Western blotting with the mitochon-
The NMR of KSCM-5 showed H NMR (300 MHz, CDCl3)
drial marker SCC and cytosolic maker calnexin antibodies ␦ 1.36 to 1.56 (m, 6H), 1.82 to 1.92 (qt, J ϭ 7.58 Hz, 2H), 2.31
showed purity of the mitochondria used (Fig. 1E), confirming to 2.36 (m, 6H), 2.34 (s, 3H), 3.90 to 3.95 (t, J ϭ 7.64 Hz, 2H),
that the pregnenolone activity identified in the earlier exper- 7.03 to 7.05 (d, J ϭ 8.03 Hz, 1H), 7.11- 7.26 (m, 7H), 7.43 to
iments was generated from the metabolic activity of mito- 7.45 (d, J ϭ 6.85 Hz, 1H). 13C NMR (300 MHz, CDCl3) ␦ 9.1,
chondria. The steroid conversion was confirmed by mass 24.5, 25.3, 26.0, 49.0, 54.6, 56.6, 111.4, 120.3, 121.2, 122.6,
spectrometric analysis (Supplemental Fig. 1, A-E). Synthesis 126.2, 126.7, 127.0, 128.9, 129.0, 142.8, 144.4, 153.4, 161.5.
of pregnenolone was reduced by 75% with siRNA1- and 95% The calculated molecular mass for C24H29N2O2 [MϩH]ϩ [mass
with siRNA4-mediated knockdown of ␴-1 (Fig. 1C), with spectrometry (electrospray ionization)ϩ] was 377.2229, and the
quantitation of the spots (Fig. 1D). As such, the loss of pro- observed was 377.2227.
gesterone is before the ⌬4 (progesterone onwards) step of
steroid synthesis. Taken together, these results strongly sug- ␦ 1.39 to 1.57 (m, 6H), 1.82 to 1.92 (qt, J ϭ 7.14 Hz, 2H), 2.33
The NMR of KSCM-11 showed 1H NMR (300 MHz, CDCl3)

582
Marriott et al.
to 2.37 (m, 6H), 2.35 (s, 3H), 3.74 (s, 3H), 3.90 to 3.95 (t, J ϭ increase in affinity at ␴-2 (Ki ϭ 41 nM) in comparison with
7.57 Hz, 2H), 6.52 (s, 1H), 6.78 to 6.80 (d, J ϭ 8.61 Hz, 1H), KSCM-1. The exclusion of both methoxy substituents at C-5
7.12- 7.33 (m, 6H). 13C NMR (300 MHz, CDCl3) ␦ 9.2, 24.5, and C-6 of the benzofuran moiety to produce KSCM-5 re-
25.3, 26.0, 49.0, 54.6, 55.6, 56.6, 95.3, 112.3, 120.6, 122.1, sulted in significantly increased affinity at both ␴-1 (Ki ϭ 7.8
122.4, 126.6, 127.0, 129.0, 143.1, 143.6, 154.6, 159.6, 161.4. nM) and ␴-2 (Ki ϭ 16 nM) in comparison with KSCM-1 and
The molecular calculated for C25H31N2O3 [MϩH]ϩ was 407.2335, KSCM-11 (Fig. 2). However, selectivity of KSCM-5 for ␴-1
and observed was 407.2339.
over ␴-2 was only 2-fold. In summary, the dimethoxybenzo-
␴-1 Receptor Binding Assays. Pharmacological charac- furan-2-carboxamide KSCM-1 molecular structure produced
terization of the synthesized ligands were performed by ra- a selective, high-affinity ␴-1 receptor ligand.
dioligand binding assays. Of the compounds screened, three
␴-Selective Ligand Structural Changes Affects Pro-
compounds (KSCM-1, KSCM-5, and KSCM-11) showed selec- tein Expression. To understand the metabolic regulation in
tivity for ␴-1 and ␴-2 receptors. ␴-1-Selective ligands usually steroidogenic cells we compared MA-10 with the nonsteroido-
possess a primary and secondary hydrophobic site separated genic COS-1 cells. We were surprised that we did not find any
by an amine functionality. The ␴-1 receptor site displays expression of ␴-1 in COS-1 cells, although it is possible that
some bulk tolerance, so this proved to be quite amenable to the level was too low to detect or the protein was efficiently
the introduction of a benzofuran moiety to produce the gen- proteolyzed. We next evaluated the effect of the ligands on
eral molecular framework. The inclusion of methoxy sub- MA-10 cell viability. Incubation with increasing concentra-
stituents at both C-5 and C-6 of the benzofuran moiety re- tions of KSCM ligands decreased cell viability, although to
sulted in both high affinity and selectivity at the ␴-1 receptor different extents (Fig. 3). For example, 1 nM KSCM-1 pro-
preferentially. For ␴-1 receptors, the Ki of KSCM-1, KSCM-5, duced a 3.5% decrease in cell viability, whereas 100 nM led to
and KSCM-11 was 27, 7.8, and 34 nM, respectively, suggest- a 16% decrease (Fig. 3A). In the case of KSCM-5, 1 nM
ing that KSCM-5 had the maximum affinity followed by decreased cell viability by 28%, whereas 100 nM resulted in a
KSCM-1 and then KSCM-11 (Fig. 2, A-C). KSCM-1 had a Ki 74% decrease (Fig. 3B). Finally, like KSCM-1, KSCM-11 at
of 527 nM at ␴-2 receptors, which indicated selectivity for lower concentrations did not greatly affect cell viability (a de-
␴-1. Moreover, these compounds were specific for the ␴-re- crease of 3.3% with 1–5 nM); however, at 100 nM there was a
ceptors because the secondary binding assays revealed insig- 62% decrease (Fig. 3C). It seems that KSCM-11 had a greater
nificant binding to non-␴-receptors. The exclusion of a me- cytotoxic effect than KSCM-1, perhaps caused by increased
thoxy substituent at C-5 of the benzofuran moiety to affinity at the ␴-2 receptor. KSCM-5 produced the most potent
produce KSCM-11 resulted in a slightly decreased affinity decrease in cell viability. This compound differs from the other
at ␴-1 (Ki ϭ 34 nM); however, we observed a significant two ligands in that it has no methoxy groups at C-5 and C-6 of
C
2875
A
B
■ Haloperidol
Ki = 1.7 nM
▲19604
■ Haloperidol
pKi = 8.8 0.04
Ki = 1.6 nM
▲ 21046
■ Haloperidol
pKi = 8.8 0.04
Ki = 1.6 nM
▲ 21052
140
120
100
80
2875
2300
1725
1150
575
Ki = 28 nM
▼ 19613
2300
1725
1150
575
0
pKi = 8.11 0.03
Ki= 7.8 nM
pKi = 7.47 0.03
Ki= 34 nM
Ki = 1.267 nM
60
40
20
0
0
-14 -12 -10
-8
-6
-4
-14 -12 -10
-8
-6
-4
-14 -12 -10
-8
-6
-4
log [drug]
log [drug]
log [drug]
E
F
D
140
120
100
80
■ Haloperidol
Ki = 13 nM
▲19603
■ Haloperidol
pKi = 8.11 0.07
Ki = 7.8 nM
▲ 21052
pKi = 7.38 0.07
Ki= 41 nM
■ Haloperidol
pKi = 8.11 0.07
Ki = 7.8 nM
▲ 21046
2875
2300
1725
1150
575
0
2875
2300
1725
1150
575
Ki = 2.841 nM
▼ 19604
pKi = 7.79 0.07
Ki= 16 nM
Ki = 528 nM
60
40
20
0
0
-14 -12 -10
-8
-6
-4
-14 -12 -10
-8
-6
-4
-14 -12 -10
-8
-6
-4
log [drug]
log [drug]
log [drug]
Fig. 2. A to C, pharmacokinetics of KSCM ligands. Ligand binding of KSCM-1 (A), KSCM-5 (B), and KSCM-11 (C) to ␴-1 receptors. Affinities Ki (nM)
were determined in rat brain homogenate in ␴-binding buffer. ␴-1 Receptors were labeled with [3H](ϩ)- pentazocine, and haloperidol served as the
reference compound. KSCM-5 exhibited the strongest binding followed by KSCM-1 and KSCM-11. D to F, ligand binding of KSCM-1 (D), KSCM-5 (E),
and KSCM-11 (F) to ␴-2 receptors. Affinities Ki (nM) were determined in PC12 cells in ␴-binding buffer. ␴-2 Receptors were labeled with
[3H]1,3-di(2-tolyl) guanidine and haloperidol as the reference compound. KSCM-5 exhibited the strongest binding followed by KSCM-11 and then very
weak binding by KSCM-1. Data represent Ki (nM) values obtained from nonlinear regression of radioligand competition binding isotherms. Ki values
are calculated from best-fit IC50 values using the Cheng-Prusoff equation. Data presented are the mean Ϯ S.E.M. of three independent experiments.
PDSP compound 19613 (control), compound 19603 (control).

␴-1 Regulates Mitochondrial Enzymatic Activity
583
C
A
B
KSCM-1
KSCM-5
KSCM-11
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
Fig. 3. Cell viability assay of the KSCM li-
gands after incubation for 24 h at varying
concentrations in MA-10 cells. KSCM-5 (B)
exhibited the greatest toxicity followed by
KSCM-1 (A) and KSCM-11 (C). Data are the
mean Ϯ S.E.M. of at least three independent
experiments.
0
20 40 60 80 100
Ligands (nM)
0
20 40 60 80 100
Ligands (nM)
0
20 40 60 80 100
Ligands (nM)
the benzofuran ring and very high affinity at both ␴-receptors. sion increased only minimally in the presence of KSCM-5 (Fig.
A nonplanar conformation is more stable than the planar con- 4A, left). The densitometric measurement (Fig. 4B) was deter-
formation (Anderson et al., 1979), because of the stronger inter- mined comparing the ratio between the ␴-1 (Fig. 4A, left) and
action with the protein conformation and charged phospholip- ␤-actin (Fig. 4A, right). This result suggests an interaction of
ids, perhaps resulting in overall less toxicity.
the ligands on the ␴-1 receptor, which is a cytosolic protein
Expression and Steroidogenic Activity of KSCM-1, localized at the junction of the ER and mitochondria. So, we
KSCM-5, and KSCM-11. We next wanted to evaluate next asked whether these ligands had an effect on the IMM
whether KSCM ligand binding affected ␴-1 receptor expres- resident protein 3␤HSD2 (Pawlak et al., 2011b), which associ-
sion. Western analysis demonstrated that incubation of ates with the IMM without membrane integration because of
MA-10 cells for 24 h with KSCM-1 and KSCM-11 increased molten globule conformation (Pawlak et al., 2011b; Prasad et
expression of the ␴-1 receptor (Fig. 4A, left), whereas expres- al., 2012). Western blotting with antibodies directed against
A
B
Ligands (nM)
Ligands (nM)
Ligands (nM)
6
4
2
0
C
KSCM-1
42
42
KSCM-1
KSCM-5
42
KSCM-1
KSCM-5
KSCM-11
29
KSCM-1
KSCM-5
KSCM-11
100
10 50
1
5
MA-10 + KSCM-1 (nM)
42
42
29
29
6
4
2
0
KSCM-5
KSCM-11
42
3βHSD2 Ab
Sigma-1 Ab
β-Actin Ab
5
10 50 100
1
MA-10 + KSCM-5 (nM)
6
4
2
0
KSCM-11
D
5
10 50 100
1
E
MA-10 + KSCM-11 (nM)
25
20
15
10
5
Prog
Preg
0
KSCM-1
(nM)
KSCM-5 KSCM-11
Origin
Mito
(nM)
(nM)
+ +
+
+
+
+ + + +
+
+
+
+NAD
+ MA-10 Mito
Fig. 4. Effect of different KSCM ligands on the ␴-1 receptor expression. KSCM ligands were added to MA10 cells at concentrations ranging from 1 to 100
nM for 24 h. A, left, Western blots showed that expression of the ␴-1 receptor increased with increasing the concentrations of KSCM-1 (top), moderately
increased with increasing concentrations of KSCM-11 (bottom), but remained unchanged with KSCM-5 (middle). Right, Western blot of the same membranes
probing with ␤-actin corresponding to each ligand. B, densitometric estimation of the Western blots in A illustrates the difference in expression with
increasing concentrations of KSCM ligands. C, effect of incubating cells with KSCM-1, KSCM-5, and KSCM-11 on the expression of the inner mitochondrial
steroidogenic enzyme 3-␤HSD2. D, mitochondrial metabolic conversion of [3H]pregnenolone to progesterone after the addition of NAD in the presence of
various concentrations of KSCM-1, KSCM-5, and KSCM-11. E, quantitative estimation of the metabolic conversion from D, which shows 27% enhanced
increase in activity in the presence of KSCM-1 at 10 nM. Data presented in B and E are the mean Ϯ S.E.M. of three independent experiments.

584
Marriott et al.
with human 3␤HSD2 (Prasad et al., 2012) and ␤-actin revealed of KSCM-1 (Fig. 5C, top), whereas KSCM-5 (Fig. 5C, middle)
no change in the levels of 3␤HSD2 (Fig. 4C) and ␤-actin (Sup- and KSCM-11 (Fig. 5C, bottom) reduced VDAC2 expression.
plemental Fig. 2) levels at any concentrations with any of the
ligands, suggesting that the KSCM series of ligands specifically independent of VDAC1. To address this, we examined the
targeted the cytosolic ␴-1 receptor. expression of ␴-1 receptor in the presence and absence of
We next asked whether the effects seen with ␴-1 were
Given the potential role of ␴-1 in steroidogenesis, we also KSCM ligands in our previously developed VDAC1 knock-
performed conversion assays in the presence of ligands. The down MA-10 cells (Bose et al., 2008). For accurate compari-
results (Fig. 4D) showed that 10 nM KSCM-1 increased proges- son we probed the same membrane with ␤-actin and ␴-1
terone conversion more than 25% (Fig. 4E), but, with further antibody independently (Fig. 5, D-F). The results showed
increases in ligand concentration, reduced conversion to the (Fig. 5, D-F, top) that, in the absence of VDAC1, ␴-1 receptor
basal level seen without KSCM-1. At 10 nM KSCM-5 and and ␤-actin (Fig. 5, D-F, bottom) expression remained un-
KSCM-11 had no effect, but conversion decreased signifi- changed compared with the parental cells. Moreover, the
cantly at higher concentrations. The possibility exists that addition of KSCM ligands did not have any significant effect
the ␴-1 receptor transiently interacts with the OMM-asso- on ␴-1 expression, confirming that the ␴-1 receptor func-
ciated porins. As such, the increased levels of ␴-1 receptor tioned independently of VDAC1.
with KSCM-1 may enhance the transport of substrate cho-
␴-1 Associates with VDAC via StAR. StAR is active at
lesterol into the mitochondria, thereby increasing proges- the OMM in a partially open conformation (Bose et al., 1999,
terone synthesis. The reduced metabolic activity seen with 2009a,b) and functions to transport cholesterol into the mi-
KSCM-5 and KSCM-11 may possibly be caused by an al- tochondria. Knowing this, our next objective was to deter-
teration in cholesterol import or an unknown mechanism. mine the role of KSCM ligands on the expression of StAR in
Role of VDAC in ␴-1 Receptor Action. We had shown MA-10 cells. In all the cases, we observed both the 37-kDa
previously that steroid synthesis requires VDAC1, because it unimported and the 30-kDa mitochondrial-imported, mature
contributes to the appropriate folding of the StAR, the protein protein, but not the 32-kDa transient intermediate protein
responsible for cholesterol fostering from the OMM to IMM (Fig. 6A). We probed the same membrane first with StAR
(Bose et al., 2008). Nevertheless, treatment with the KSCM (Fig. 6A) antibody and then with ␤-actin antibody (Supple-
ligands did not alter VDAC1 expression levels (Fig. 5A) com- mental Fig. 4A) independently. We were surprised to find
pared with ␤-actin (Supplemental Fig. 3), suggesting that the that KSCM-1, but not KSCM-5 or KSCM-11 (Fig. 6A, middle
ligands have no effect on mitochondrial-associated protein and bottom), increased the expression of unimported 37-kDa
expression. However, next we probed with VDAC2 antibody StAR (Fig. 6A).
(Fig. 5B, left) and compared with ␤-actin (Fig. 5B, right)
We next performed coimmunoprecipitation experiments to
expression of the same membrane. The densitometric analy- determine whether there was a direct interaction between
sis (Fig. 5C) showed increased expression after the addition ␴-1 receptor and StAR or VDAC2. Coimmunoprecipitation of
C
Ligands (nM)
Ligands (nM)
Ligands (nM)
6
4
2
0
B
A
KSCM-1
42
42
42
KSCM-1
KSCM-1
22
22
32
32
KSCM-1
KSCM-5
KSCM-11
100
10 50
MA-10 + KSCM-1 (nM)
1
5
KSCM-5
KSCM-5
6
4
2
0
KSCM-5
KSCM-11
22
KSCM-11
32
β-Actin Ab
VDAC2 Ab
VDAC1 Ab
5
10 50 100
1
MA-10 + KSCM-5 (nM)
6
4
2
0
KSCM-11
F
D
E
KSCM-11 (nM)
KSCM-1 (nM)
KSCM-5 (nM)
5
10 50 100
1
Sigma-1 Ab
29
42
29
Sigma-1 Ab
Sigma-1 Ab
β-Actin Ab
29
42
MA-10 + KSCM-11 (nM)
β-Actin Ab
42
β-Actin Ab
Fig. 5. Effect of ␴-1 receptor on the outer mitochondrial proteins. A, effect of increasing concentrations of KSCM-1, KSCM-5, and KSCM-11 on VDAC1
expression as determined by Western blotting. B, left, effect of increasing concentrations of KSCM-1 (top), KSCM-5 (middle), and KSCM-11(bottom)
on VDAC2 expression as determined by Western blotting. Right, the expression of ␤-actin when probed the same membrane presented at left under
identical conditions. C, densitometric estimation of the effect of KSCM ligands on VDAC2 from B, left expression compared with ␤-actin in B, right.
VDAC2 expression was increased with KSCM-1 up to 50 nM, and then partially decreased. KSCM-5 reduced expression at 100 nM, and KSCM-11
reduced expression at 5 nM. D, effect of KSCM-1 on ␴-1 receptor expression in VDAC1 knockdown cells. Western blot with ␴-1 antibody showed
increased expression after addition of KSCM-1 at any concentration, except 100 nM. E and F, ␴-1 expression in VDAC1 knockdown cells incubated
with KSCM-5 and KSCM-11 ranging from 1 to 100 nM. Western blots indicated that ␴-1 expression remained unchanged in VDAC1 knockdown cells.
Unaffected expression of ␤-actin is presented (bottom).

␴-1 Regulates Mitochondrial Enzymatic Activity
585
B
A
Ligands (nM)
29
37
30
Sigma-1 Ab
KSCM-1
KSCM-5
37
30
37
30
StAR Ab
37
30
23
Tim23 Ab
KSCM-11
StAR Ab
Fig. 6. Expression of StAR and its interaction with proteins involved in steroidogenesis. A, expression showing the unchanged expression of
mitochondrial unimported 37-kDa and imported 30-kDa StAR after incubation with KSCM-5 and KSCM-11. KSCM-1, at or above 10 nM, stabilizes
the expression of unimported StAR, but imported 30-kDa StAR expression was unaffected. B, coimmunoprecipitation of the MA-10 mitochondria with
the indicated antibodies followed by Western blotting with ␴-1 antibody (top) and StAR antibody (middle). The experiment shows the interaction of
StAR with ␴-1. Western blotting of the same coimmunoprecipitation samples with IMM resident protein Tim23 (bottom) antibody showed that Tim23
was present in MA-10 cells and pig adrenals; it did not interact with the other proteins.
the digitonin lysate from the mitochondria isolated from show the reverse stereoselectivity with a lower-affinity
MA-10 cells showed that VDAC2 and ␴-1 antibodies pulled range (Hellewell et al., 1994). The ␴-1-receptor sequence
down the ␴-1 receptor (Fig. 6B, top). StAR is a 37-kDa protein contains a 22-amino acid retention signal for the endoplas-
(Bose et al., 2008) and thus it is higher than the 28-kDa ␴-1 mic reticulum at its N-terminal region and the two short
(Fig. 6A), confirming that StAR interaction with the ␴-1 C-terminal hydrophobic amino acid sequences that may be
receptor. Probing the same membrane with the Tim23 anti-
body failed to show any interaction with the StAR or the ␴-1
receptor (Fig. 6B, bottom), suggesting that the StAR interac-
tion occurred before import into the mitochondria. Similar
Western blotting of the flow-through from the previously
mentioned coimmunoprecipitation experiments (Supplemen-
tal Fig. 4B) with ␴-1 antibody showed minimal intensity
when pulled down with VDAC2, which led us to conclude that
the maximal interaction was between ␴-1 and VDAC2 (Fig.
6B). Because StAR is expressed on hormonal stimulation in
the adrenal and gonads, we conclude that StAR acts as a
bridge between ␴-1 and VDAC2 for cholesterol loading onto
the OMM.
involved in sterol binding (Hanner et al., 1996).
Steroid hormones are not stored in the tissue for future
use. Rather, the steriodogenic tissues, including adrenals,
gonads or ovaries, and brain, generate steroids acutely, when
the need arises. This requires the transport of a huge amount
of cholesterol from the cytoplasm into the mitochondria,
where it serves as the sole substrate for the catalysis of
pregnenolone, the first steroid in the pathway. However, the
mechanism of enhanced cholesterol transport remains un-
known. Studies have shown that the interaction of VDAC
with StAR, a protein that acts exclusively on the OMM
before import (Bose et al., 2002), enhances cholesterol
import, and this requires StAR to undergo a structural
transition to exert activity (Miller and Bose, 2011). In
addition to the VDAC found at the OMM, the plasma
membrane of various cell types contain this protein,
termed plVDAC (Dermietzel et al., 1994; Ba`thori et al.,
1999; Buettner et al., 2000; Bahamonde et al., 2003;
Elinder et al., 2005; Akanda and Elinder, 2006). The dif-
ferential targeting is probably caused by an alternative
first exon in the murine VDAC1 gene that codes a leader
peptide at its N terminus (Buettner et al., 2000). This
peptide serves as a signal to target the protein to the
plasma membrane via the Golgi apparatus, and it is even-
tually cleaved away to produce the plVDAC protein. Be-
cause the N-terminal segment of VDAC is involved in
voltage gating, it may adopt different conformations de-
pending on the factors external to the protein (Ujwal et al.,
2008). Using electron microscopy and NMR studies with a
nanodisc technology, it has been demonstrated that the
presence of phospholipid membrane facilitates this target-
Discussion
A key criterion used to identify ␴-receptors is their abil-
ity to bind several chemically unrelated drugs with high
affinity. These drugs include psychotomimetic benzomor-
phans, 1-(1-phenylcyclohexyl)piperidine (phencyclidine)
and its derivatives, cocaine and its derivatives, ampheta-
mine, certain neuroleptics, many atypical antipsychotic
agents, anticonvulsants, cytochrome P450 inhibitors,
monoamine oxidase inhibitors, histaminergic receptor li-
gands, peptide from the neuropeptide Y or calcitonin gene-
related peptide families, substance P, and neuroactive ste-
roids. The ␴-binding sites could be labeled by various
specific radioligands, including [3H]2S-[2␣,6␣,11R*]-
1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-
methano-3-benzazocin-8-ol (SKF-10,047), [3H](ϩ)3-(3-
hydroxyphenyl)-N-(1-propyl)-piperidine, [3H]haloperidol,
[3H]1,3-di-ortho-tolylguanidine, and [3H](ϩ) pentazocine. ing to or interaction with different proteins (Raschle et al.,
Pharmacological structure/activity studies led to the defi- 2009). Because VDAC1 knockout mice are viable (Anflous et al.,
nition of two subclasses of ␴ sites, ␴-1 and ␴-2 (Quirion et 2001), it is likely that additional mechanisms promote choles-
al., 1992). The two sites were distinguished based on their terol transport, possibly through the interaction of the ␴-1 re-
different drug selectivity patterns and molecular weights. ceptor associated with VDAC2 at the MAM region. Because a
The ␴-1 site shows a stereoselectivity with high affinity for specific region of MAM anchors to the OMM, where the ␴-1
dextrogyre isomers of benzomorphans, whereas ␴-2 sites receptor plays a crucial role for maintaining connectivity, we

586
Marriott et al.
Thinnes FP, Fishman GI, and Spray DC (1994) Cloning and in situ localization of
a brain-derived porin that constitutes a large-conductance anion channel in astro-
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Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, and Ceccatelli
S (2005) Opening of plasma membrane voltage-dependent anion channels (VDAC)
precedes caspase activation in neuronal apoptosis induced by toxic stimuli. Cell
Death Differ 12:1134–1140.
Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, and Ruoho AE
(2009) The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous ␴-1
receptor regulator. Science 323:934–937.
suggest that VDAC2 may play a greater role than VDAC1 in
mediating this connection. Because VDAC2 is expressed mostly
with the presence of ␴-1 receptor binding ligand KSCM-1, we
propose that deletion of ␴-1 receptor disrupts the bridge formed
by VDAC2, resulting in an inhibition of cholesterol influx into
the mitochondria.
Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kempner E, and
Glossmann H (1996) Purification, molecular cloning, and expression of the mam-
malian sigma1-binding site. Proc Natl Acad Sci U S A 93:8072–8077.
Hayashi T, Rizzuto R, Hajnoczky G, and Su TP (2009) MAM: more than just a
housekeeper. Trends Cell Biol 19:81–88.
Hayashi T and Su TP (2001) Regulating ankyrin dynamics: roles of ␴-1 receptors.
Proc Natl Acad Sci U S A 98:491–496.
Acknowledgments
Ki determinations and receptor binding profiles were generously
provided by the National Institute of Mental Health’s Psychoactive
Drug Screening Program, Contract HHSN-271-2008-00025C, di-
rected by Dr. Bryan L. Roth at the University of North Carolina
(Chapel Hill, NC) and Project Officer Jamie Driscol at the National
Institute of Mental Health (Bethesda, MD).
Hayashi T and Su TP (2003) Intracellular dynamics of ␴-1 receptors (␴1 binding
sites) in NG108–15 cells. J Pharmacol Exp Ther 306:726–733.
Hayashi T and Su TP (2007) ␴-1 receptor chaperones at the ER-mitochondrion
interface regulate Ca2ϩ signaling and cell survival. Cell 131:596–610.
Hellewell SB, Bruce A, Feinstein G, Orringer J, Williams W, and Bowen WD (1994)
Rat liver and kidney contain high densities of ␴1 and ␴2 receptors: characteriza-
tion by ligand binding and photoaffinity labeling. Eur J Pharmacol 268:9–18.
Jbilo O, Vidal H, Paul R, De Nys N, Bensaid M, Silve S, Carayon P, Davi D, Galie`gue
S, Bourrie´ B, et al. (1997) Purification and characterization of the human SR
31747A-binding protein. A nuclear membrane protein related to yeast sterol
isomerase. J Biol Chem 272:27107–27115.
Authorship Contributions
Participated in research design: Marriott, Prasad, and Bose.
Conducted experiments: Marriott, Prasad, and Thapliyal.
Performed data analysis: Marriott, Prasad, and Bose.
Wrote or contributed to the writing of the manuscript: Marriott,
Prasad, and Bose.
Kakkar T, Pak Y, and Mayersohn M (2000) Evaluation of a minimal experimental
design for determination of enzyme kinetic parameters and inhibition mechanism.
J Pharmacol Exp Ther 293:861–869.
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Address correspondence to. Dr. Himangshu S. Bose, Department of Biochem-
istry, Mercer University School of Medicine and Memorial University Medical Center,
Anderson Cancer Research Institute, Hoskins Research Building, Savannah, GA
31404. E-mail: bose_hs@mercer.edu or bosehi1@memorialhealth.com