The Σ1 Receptor Engages the Redox-Regulated HINT1 Protein to Bring Opioid

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Original Research Communication

The σ1 receptor engages the redox-regulated HINT1 protein to bring opioid

analgesia under NMDA receptor negative control

María Rodríguez-Muñoz1, Pilar Sánchez-Blázquez1, Raquel Herrero-Labrador1, Ricardo

Martínez-Murillo1, Manuel Merlos2, José Miguel Vela2, Javier Garzón1

1Cajal Institute, Consejo Superior de Investigaciones Científicas (CSIC), Avenida

Doctor Arce, 37. 28002 Madrid, Spain. 2Drug Discovery & Preclinical Development,

Esteve. Scientific Park of Barcelona, Bardiri y Reixac 4-8, 08028, Barcelona, Spain.

Running head: σ1R engages NMDAR control on MOR

Correspondence to: Javier Garzón. Neurofarmacología, Instituto Cajal, Avenida

Doctor Arce 37, 28002 Madrid, Spain. Tel: 34 91 5854733, Fax: 34 91 5854754. E-

mail: [email protected]

Antioxidants & Redox Signaling

Word count: 6950

Reference number: 70

Grayscale Illustrations: Figures 5, 6, 8 and 9

Color Illustrations: Figures 3 and 4

Color online, B&W print: Figures 1, 2, 7, and 10 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 1

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Abstract

Aims: The in vivo pharmacology of the sigma 1 receptor (σ1R) is certainly complex;

however, σ1R antagonists are of therapeutic interest because they enhance mu-opioid

receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we

investigated whether the σ1R is involved in the negative control that glutamate N-

methyl-D-aspartate receptors (NMDARs) exert on opioid antinociception. Results: A

redox-regulated process situates MOR signaling under NMDAR control, and in this

context, the σ1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit.

The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1)

coupled to the regulator of G-protein signaling RGSZ2-neural nitric oxide synthase

(nNOS) assembly. Activated MORs stimulate the production of NO, and the redox zinc

switch RGSZ2 converts this signal into free zinc ions that are required to recruit the

redox sensor PKCγ to HINT1 proteins. Then, PKCγ impairs HINT1-RGSZ2 association

and enables σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid

signaling. The inhibition of NOS or the absence of σ1Rs prevents HINT1-PKCγ

Antioxidants & Redox Signaling interaction and MOR-NMDAR cross-regulation fails. The σ1R antagonists transitorily

remove the binding of σ1Rs to NR1 subunits, facilitate the entrance of negative

regulators of NMDARs, likely Ca2+-CaM, and prevent NR1 interaction with HINT1,

thereby impairing the negative feedback of glutamate on opioid analgesia. Conclusion:

The σ1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from

the negative influence of NMDARs, and they also reset antinociception in morphine

tolerant animals. Moreover, σ1R antagonists alleviate neuropathic pain, probably by

driving the inhibition of up-regulated NMDARs. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 2

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Introduction

The Mu-opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that selectively

controls the perception of nociceptive sensorial signals. Unfortunately, the frequent

administration of opioids such as morphine and derivatives typically leads to the

development of analgesic tolerance, promoting little recycling/resensitization of their

receptors (12) and other adaptive processes that result in MOR desensitization on the

cell surface (14). In animals, tolerance to the antinociceptive effects of opioids can be

observed even after a single and adequate dose, and thus, morphine can induce acute

strong tolerance via the N-methyl-D-aspartate acid glutamate receptor (NMDAR)/neural

nitric oxide synthase (nNOS)/zinc metabolism (45; 47). In this scenario, the physical

association between MORs and NMDARs within a specialized protein assembly

facilitates their functional cross-regulation (49).

The sigma 1 receptor (σ1R) has been proposed as a tonic anti-opioid system (39)

that modulates the activity-induced sensitization in nociceptive pathways (8). The σ1Rs

are widely expressed in nervous tissue, presenting high levels in areas that are

Antioxidants & Redox Signaling associated with pain control (28). Whereas σ1R agonists facilitate nociception (27; 69),

σ1R antagonists reduce the allodynia and hyperalgesia that accompany neuropathy in

different animal models, improving the activity of opioids against nociceptive stimuli

(8; 52; 53; 70). The σ1R was initially considered to be a type of opioid receptor (35);

however, the σ1R lacks glycosylation, and its molecular structure suggests a different

class of regulatory function, most likely that of chaperones (21). The σ1R constitutes a

unique class of linear proteins that only has two transmembrane domains (3), with both

N and C terminal sequences projecting to the same side, cytosol (59) or extracellular

space (4), similar to the hairpin-like structure of caveolins, which are non-neural The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) scaffold proteins (42). This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 3

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The σ1R activity is modulated through a series of endogenous and exogenous

substances. The pharmacology of the σ1R is complex, with exogenous ligands showing

different profiles depending on the system under study (38). Notwithstanding this

drawback, σ1R ligands are of therapeutic interest for the treatment of neurological

diseases (31), substance abuse syndromes (46) and NMDAR-related neuropsychiatric

disorders (22) or as adjuvants of opioid analgesia (25; 39; 64). According to the anti-

opioid function of the σ1R (39), σ1R antagonists enhance the analgesic effect of

systemic morphine, which is prevented by σ1R agonists, and also restore morphine

-/- analgesia in tolerant mice (64). As expected, σ1R mice exhibit an increased response

to morphine antinociception that cannot be regulated by σ1R ligands (57). Importantly,

the opioid effects that are enhanced by σ1R antagonists are those regulated by the

NMDAR/NOS/CaMKII pathway (70); thus, σ1R ligands do not modify morphine-

induced hyperlocomotion or gastrointestinal transit inhibition. The positive features of

the highly selective σ1R antagonist S1RA makes this drug a good candidate for the

treatment of neuropathic pain (53), and this treatment has satisfactorily completed phase Antioxidants & Redox Signaling I safety and pharmacokinetic evaluation in humans (1).

The σ1R ligands modulate NMDAR functions in vivo and in vitro (36; 41; 55).

Indeed, in cellular expression systems and in vitro assays, the σ1R displays calcium-

dependent binding with NMDAR NR1 subunits (55). Because σ1Rs also associate with

MORs (25), it is possible that these proteins regulate opioid function within the protein

assembly that, via the histidine triad nucleotide-binding (HINT1) protein, redox

signaling and zinc metabolism, support the MOR-NMDAR physical association and

functional cross-regulation (48-50).

Within this background, this study analyzed the potential role of σ1Rs in the The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

cross-regulation between MORs and NMDARs in the mesencephalic periaqueductal This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 4

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grey matter (PAG), a supraspinal region that regulates spinal nociceptive signals. The

σ1Rs associate with NMDAR NR1 subunits and σ1R antagonists promote the binding

of negative regulators of NMDAR activity. The negative feedback that NMDARs

display on MOR signaling requires the NO- and zinc-dependent recruitment of PKCγ to

the HINT1 proteins followed by σ1R-NR1 binding to the MOR-HINT1 complex.

Consequently, σ1R antagonists uncouple NMDAR effects from MOR signaling, thereby

enhancing morphine analgesia and reducing the development of opioid tolerance. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 5

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Results

Organization of the σ1R receptor in the cell membrane

In the nervous tissue, the σ1R exists as long and short isoforms (21; 58). The long form

of σ1R comprises 223 amino acid residues, while the short form contains 106 amino

acids. The σ1R has a hairpin structure with a short N terminal sequence before the first

hydrophobic transmembrane domain that leads to the loop region, followed by the

second transmembrane domain and the long C-terminal domain (cd). The short form of

σ1R contains a clipped C-terminal sequence that differs from that of the long form in

the four last residues. There is some controversy regarding the potential arrangement of

σ1R in the plasma membrane, specifically with respect to whether both the N- and C-

terminal sequences are directed to the cytosolic side leaving the loop in the extracellular

milieu, or in the opposite orientation with the loop facing the cytosolic side (4; 44) (Fig.

1A).

The σ1R in the ER membrane binds Bip in a calcium-dependent manner, and

this binding is retained through the cd region (residues 112-223; σ1R) (43). In cell

Antioxidants & Redox Signaling expression systems, σ1R shows calcium-dependent interaction with the NR1 subunit of

NMDAR, and both proteins can be co-immunoprecipitated from brain synaptosomes (4;

55). In in vitro assays, σ1R binds to the NR1 cytosolic C-terminal region C0-C1-C2

(55). The σ1R loop has a SIM domain (LIVEL: 61-65) that is typical of intracellular

interactions (Fig. 1A and Supplemental Fig. 1), and the long σ1R binds to the NMDAR

NR1 subunit via fluctuations in calcium levels, such as those that are observed in the

cytosol (55). Thus, at least a part of the C-terminal region should be located on the

cytosolic side. Indeed, the ligand-binding pocket of the σ1R comprises the two TM

domains and the hydrophobic region of the σ1Rcd (44), containing a potential The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) membrane attachment amino acid sequence (43). SBDLI and SBDLII (steroid binding- This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 6

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like domains) are located in the σ1R TM2 and cd regions, and whereas the average

charge of SBDLI is mostly neutral, that of SBDLII is negative, offering the potential for

calcium regulation (Fig. 1B). The short σ1R displayed calcium-independent binding to

NR1 C0-C1-C2; however, this binding in the long isoform was calcium-dependent (Fig.

1C), suggesting that the cd impairs the binding of the long σ1R form to the NR1 subunit

and that calcium eliminates this barrier. A tentative model for σ1R arrangement is

presented in Fig. 1D.

Concerning the arrangement of σ1R in the cell membrane, we examined the

calcium-dependent binding of σ1Rs to NR1 subunits through peptide interference (Fig.

2 and Supplemental Fig. 2). This approach suggested that NR1 C0 (839-853) and NR1

C1 (864-878) bind to the σ1R. Peptide mapping of the hydrophobic regions in the NR1

C0 and C1 segments greatly enhanced the interaction between the σ1Rs and NR1

subunits, suggesting that both of the NR1 hydrophobic domains interact and that σ1R

disrupts this interaction to achieve NR1 binding. It is therefore possible that peptide 9

interferes with σ1R binding in the NR1 region of the 883 residue; however, this

Antioxidants & Redox Signaling interference could be masked by peptide 9 better exposing the NR1 C0-C1 domain for

interaction with third part proteins. Peptide interference indicated that two regions of the

σ1R loop could be implicated in its interaction with NR1 subunits; the first region lies

between residues 40 and 60, and the second lies between residues 71 and 80. Mapping

the σ1R cdomain (174-223) indicated that the HR1 region of NR1 C0 does not interact

with σ1R 184-203 sequence, and then the NR1 C0 region that is most likely involved in

σ1R binding is 839-848. Notably, peptides mapping the σ1R cd hydrophobic regions H3

and H4 impaired the binding of this receptor to the NR1 subunit. The interaction of

these hydrophobic regions with the TM1 and TM2 domains is essential to maintain the The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) conformation of the σ1R as able to bind to the NR1 C0-C1 domains (43; 44). Thus, an This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 7

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excess of these peptides would remove the σ1R H2 and H3 regions from the TM

domains, disorganizing the structure of the σ1R and consequently reducing its

interaction with the NR1 subunit (Supplemental Fig. 2).

A similar approach revealed that the HINT1-binding region on NR1 C0-C1

overlaps with the σ1R-binding region. Thus, peptides mapping to the NR1 C0

hydrophobic region enhanced the HINT1-NR1 interaction. However, peptides mapping

to the corresponding region in the NR1 C1 segment diminished this interaction. These

observations indicate that HINT1 unfolds the NR1 but primarily binds to the NR1 C1

hydrophobic region, ignoring the hydrophobic region on the NR1 C0 segment. Whereas

in the absence of peptides 4 and 10, the σ1R and HINT1 proteins exhibited noticeable

binding to NR1 C0-C1, the binding of Ca2+-CaM was certainly weak. It is possible that

the disruption of the NR1 intrahydrophobic interaction through Ca2+-CaM is weaker

than that achieved by σ1R or HINT1; thus, the Ca2+-CaM binding to NR1 subunits

likely requires the assistance of third-party proteins. This possibility has been

previously suggested through the binding of the cytoskeletal protein α-actinin2 with the

Antioxidants & Redox Signaling C0 region of the NR1 subunits (40). The NR1 subunit contains two putative regulatory

sites for Ca2+-CaM, with one site in the C0 segment and the other in the C1 region (11),

and the disruption of the NR1 C0-C1 hydrophobic interaction through either peptide 4

or 10 exposed one of the NR1 sites for Ca2+-CaM binding. Accordingly, the presence of

both peptides eliminated Ca2+-CaM binding to the NR1 subunits (Fig. 2).

The juxtaposition of the σ1R loop and NR1 C0-C1 regions revealed

complementary charges between the loop emerging from TM1 and the upper half of the

NR1 C1 segment, followed by putative hydrophobic interactions between the SIM-

containing sequence 58-66 and the NR1 hydrophobic region S890 (Fig. 3A). In The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) addition, the pre-incubation of SUMO1 with σ1R impaired subsequent binding to the This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 8

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NR1 subunits, and SUMO1 weakly disrupted the existing σ1R-NR1 association (Fig.

3B). Thus, the SIM-containing loop is involved in σ1R binding to NR1, and this region

is hardly available during their association, reflecting the interaction of the SIM-

associated negative region with complementary charges in the NR1 C1 segment,

subsequently weakening the possible SUMO-SIM interaction (24).

Considering our (Fig. 3C) and other (4; 55) data, we constructed the model of the σ1R-

NR1 interaction that is shown in Fig. 3D, in which σ1R is arranged in the plasma

membrane with the SIM-containing loop facing the cytosol and the N-terminal sequence

facing the extracellular space. The orientation of the short C-terminal sequence that is

outside of the TM2 is tentatively orientated to the extracellular space; however, this

sequence could also be facing the cytosolic side. The region of the σ1Rcd that is

potentially regulated by calcium (SBDLII) is located on the cytosolic surface of the cell

membrane. Thus, our arrangement of the σ1R in its interaction with the NMDAR NR1

subunit maintains the essential described features for σ1Rs in the cell membrane (4; 44)

but with the cd and loop hydrophobic regions, which show complementary charges,

Antioxidants & Redox Signaling situated on the cytosolic side of the membrane. In the absence of calcium, the

interaction between the σ1R and the NR1 subunit is occluded. The positive calcium ions

bind to the hydrophobic region of the σ1Rcd and neutralize the negative charge that is

required to bind the SIM-containing positive region of the loop. Thus, the calcium-

bound σ1Rcd region can now disrupt the internal hydrophobic interaction NR1 C0

(HR1)-NR1 C1 (HR2), exposing the required binding surface to the σ1R loop. We

could detect these signaling proteins in mouse PAG neural cells using antibodies against

the MOR, σ1R and NMDAR NR1 subunits. Immunohistochemical analysis revealed a

high degree of co-localization in the lateral PAG between the NR1 subunit and its The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) regulator, the σ1R. In this region, the MOR showed a discrete co-localization with these This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 9

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proteins. The triple co-localization of the MOR, σ1R and NR1 subunits could be

observed as a series of white spots located at the cell periphery and also in fibers. This

distribution/co-localization is compatible with σ1Rs regulating MOR and NMDAR

function (Fig. 4 and Supplemental Fig. 3).

These observations situate σ1R in the protein assembly that controls MOR

activity via NMDARs. Thus, we addressed whether the antinociception and molecular

changes that are produced by morphine in the presence of σ1R antagonists fit this

model.

Antagonists of σ1Rs enhance morphine-evoked supraspinal antinociception

The MOR in the mesencephalic PAG plays the most relevant role in the antinociception

produced by opioids when injected by the intracerebroventricular (icv) route. The icv

administration of all the substances studied circumvents the possibility that the drugs

reach receptors beyond the brain. Subsequently, the capacity of morphine to produce

supraspinal antinociception, and that of the studied drugs to modulate this effect, was

Antioxidants & Redox Signaling assessed through the warm water tail flick test (see Methods). The distribution of the

thermal stimulus over two thirds of the tail and the cut-off of 10 s protect the mice from

tissue damage, as well as preventing the particular thermal sensitivity of a discrete point

of the tail from compromising the data. Moreover, the mice practically do not anticipate

their responses in using this analgesic test when they are studied several times during a

time-course study.

The icv administration of the highly selective σ1R antagonist S1RA [E-52862;

(53)] 10 min or 30 min before morphine treatment (3 nmol, icv) increased the analgesic

activity when evaluated 30 min post-opioid injection. S1RA given 1 h or longer before The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) morphine produced no such effect. Therefore, 30 min was selected to study the effect of This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 10

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σ1R ligands on the capacity of morphine to promote supraspinal analgesia in the

thermal tail-flick test. We have reported a model for the detection of σ1R antagonist

activity, and S1RA is likely the most potent and pure σ1R antagonist available (55).

S1RA at 3 nmol increased the analgesic activity of morphine; other antagonists, such as

BD1047 and NE100, also performed well in this paradigm, whereas BD1063 weakly

increased morphine analgesia. The agonist PRE084 did not affect morphine analgesia

but prevented S1RA from enhancing opioid antinociception (Fig. 5A).

Neurosteroids, putative endogenous regulators of the σ1R, were also studied.

Pregnenolone is a σ1R agonist, and its metabolite progesterone is a σ1R antagonist.

Pregnenolone and its acetate derivative enhance morphine antinociception; however, the

sulfate form of pregnenolone does not show this effect. Progesterone enhances

morphine antinociception, an effect that is reduced by pregnenolone sulfate (Fig. 5B),

suggesting that pregnenolone, but not its sulfate form, is rapidly converted into

progesterone.

Antioxidants & Redox Signaling Morphine increases NMDAR activity: effect of σ1R antagonism

At the molecular level, the icv administration of 10 nmol morphine increased the

function of the NMDAR-CaMKII pathway. This opioid increased the phosphorylation

of NR1 subunits (PKC on S890) and NR2 subunits (Src on Y1325). Moreover,

morphine increased the T286 autophosphorylation of the Ca2+- and CaM-dependent

serine and threonine kinase CaMKII, an NMDAR effector. As a result of the MOR-

induced activation of NMDARs, the MOR-NR1 association was weakened, and these

changes persisted beyond the morphine analgesic time-course (49). The presence of 3

nmol S1RA, administered 30 min before morphine treatment, nearly abolished CaMKII The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) activation, reduced NR1/NR2 phosphorylation and preserved the MOR-NR1 inhibitory This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 11

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association during the intervals that corresponded to the morphine analgesic time-course

and beyond (Fig. 5C and Supplemental Fig. 4).

The capacity of S1RA to enhance morphine antinociception or alter MOR-

induced NMDAR changes decreased as the S1RA-morphine interval increased (Fig.

6A). The σ1R antagonists S1RA, BD1047 and, to a minor extent BD1063, when given

before the first dose of morphine (priming dose), protected the analgesic effects of a

subsequent dose of morphine (test dose) against acute tolerance (Fig. 6B). In this

paradigm, the S1RA-morphine intervals of 30 min and 1 h were more successful than

that of 10 min. S1RA icv-injected between 1 h and 24 h before morphine neither

enhanced morphine analgesia nor prevented the activation of MOR-coupled NMDARs

(Fig. 5A & 6A); however, the σ1R antagonist still moderately decreased the

antinociceptive acute tolerance (Fig. 6B). In a previous study, systemic S1RA was

found to restore morphine analgesia in mice rendered tolerant by repeated subcutaneous

administration of this opioid (64). Thus, we addressed whether S1RA was capable of

such positive effects on mice that had received a daily icv dose of 10 nmol morphine for

Antioxidants & Redox Signaling 7 days (Fig. 6C). Antinociception was evaluated 30 min after the delivery of each

morphine dose. The analgesic response diminished during the chronic morphine

protocol, yet on day 7 the icv administration of 3 nmol S1RA 20 min before morphine

enabled the opioid to produce an analgesic effect comparable to that of the first 10 nmol

dose delivered on day 0 of the assay. At the molecular level, chronic morphine

weakened the association of MORs with NMDAR NR1 subunits, whereas it enhanced

the PKC-mediated phosphorylation of NR1 serine 890 and the activating

autophosphorylation of CaMKII. These changes revealed an increase in NMDAR

activity that returned to control levels when the chronic morphine tolerant mice received The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 12

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a single icv dose of S1RA (Fig. 6C). Thus, the rescue of morphine analgesia from

tolerance correlated with the deactivation of NMDARs.

The σ1Rs controls the interaction of NR1 subunits with NMDAR inhibitors and

the redox-regulated HINT1 protein

The MOR C terminus interacts with HINT1, which carries the regulator of the G-

protein signaling RGSZ2-neural nitric oxide synthase (nNOS) complex (2; 15; 20). The

RGSZ2 binds to the HINT1 protein in a zinc-independent manner and prevents the

entrance of NMDAR NR1 subunits. Thus, activated MORs release the RGSZ2 negative

control on nNOS and stimulate the production of NO to release zinc ions from RGSZ2

zinc domain (54) (Fig. 7A). This activity promotes the zinc-dependent binding of the

redox sensor PKCγ to HINT1 proteins through zinc ions bridging HINT1 histidines

with the cysteines of the PKCγ regulatory domain (15; 51), and both RGSZ2 and PKCγ

bind simultaneously to the redox-regulated scaffold HINT1 protein (Fig. 7A). The

MORs provide activated GαGTP subunits that bind to the RGS domain of RGSZ2,

Antioxidants & Redox Signaling exposing the HINT1 protein to the effect of PKCγ (48) (Fig. 7A). The phosphorylation

of HINT1 is the switch that releases RGSZ2, bringing NMDARs under MOR control.

Within the MOR-HINT1-σ1R-NR1 protein assembly, the NMDAR displays low

activity (63). PKCγ increases the σ1R-NR1 interaction and weakens that of HINT1 with

NR1 subunits (Fig. 7A). This concatenated process situates NMDARs close to MORs to

build together the negative feedback on opioid signaling.

The binding of the σ1R to the NR1 subunit covers a region that is critical for the

high-affinity binding of negative regulators of NMDAR function, such as HINT1 and

Ca2+-CaM (11; 63). The in vitro assays indicated that pregnenolone sulfate enhanced The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) and progesterone diminished the association of NR1 with the long isoform of the σ1R. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 13

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Upon the reduction of σ1Rs binding to NR1 subunits, the NMDAR subunit was made

available for CaM (in the presence of peptide 4 and 2.5 mM CaCl2) or HINT1 binding.

This pattern was also observed for the exogenous ligands of the σ1R receptor, and while

the antagonists S1RA and BD1047 stimulated CaM binding, the agonist PRE084 did

not (Fig. 7B). In addition, the peptide interference assay showed that σ1R cd peptides 7

and 10 could couple to the steroid-binding domain, reducing the σ1R-NR1 interaction.

It is possible that these peptides and σ1R antagonists share a common mechanism that

alters the structure of the σ1R and then diminishes its affinity for NR1 binding

(Supplemental Fig. 2). Thus, σ1R antagonists by affecting the interaction of σ1Rs with

NR1 subunits, regulate the activity of NMDARs.

The σ1R is essential for MOR-NMDAR cross-regulation

-/- In σ1R mice the opioids showed an enhanced capacity to produce antinociception,

and in these mice, the antinociceptive peak effect of an icv dose of 3 nmol morphine

was comparable to that produce by 10 nmol morphine in wild-type mice (Fig. 8A). The Antioxidants & Redox Signaling administration of an agonist of NMDARs, NMDA, to wild-type mice significantly

reduced the capacity of morphine to produce antinociception; however, NMDA failed to

-/- do so in σ1R mice. Because MOR-NMDAR cross-regulation is a redox-regulated

process that depends on NO and zinc metabolism (51; 54), in wild-type mice, the

inhibition of NOS enhanced morphine antinociception and prevented NMDA from

reducing the antinociceptive capacity of morphine; however, this approach was not

-/- effective in the σ1R mice (Fig. 8A). At the molecular level, the inhibition of NOS

prevented morphine from recruiting PKCγ in the MOR environment, and then opioid- The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) and PKCγ-triggered activation of the NMDAR-CaMKII pathway, as well as the This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 14

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weakening of MOR-NR1 interaction were not observed (Supplemental Fig. 5).

Synaptosomal membranes from mice that were treated in vivo with NOS inhibitors

responded to SNAP, NO donor, or ZnCl2, recruiting PKCγ to the MOR environment.

This effect was prevented by the co-incubation of the PAG membranes with TPEN, a

zinc chelator. These observations indicate that the in vivo administration of NOS

inhibitors specifically prevented morphine from stimulating NO production but left

operative the NO-mediated removal of zinc ions from zinc-fingers. The MOR-coupled

HINT1 protein binds PKCγ through zinc ions (54). Accordingly, in PAG synaptosomes

-/- from HINT1 mice, SNAP or zinc ions did not stimulate PKCγ arrival at the MOR.

The absence of σ1Rs greatly impaired the arrival of PKCγ to MORs, suggesting an

altered MOR-HINT1 relationship, but some response to SNAP and zinc ions could still

be observed (Fig. 8B).

The analgesic acute tolerance that an icv-dose of morphine produces in wild-

type mice can be reverted by NMDAR antagonists, PKC inhibitors and NOS inhibitors.

-/- -/- However, this effect could not be produced in σ1R mice (Fig. 8C). In σ1R mice, the Antioxidants & Redox Signaling

NMDAR-mediated regulation of MOR signaling is impaired, and this control is

-/- apparently taken beyond the receptor level, most likely at the effectors. Thus, in σ1R

mice, this protective desensitization not only affects MORs but also other GPCRs, such

as α2-adrenoceptors and cannabinoids (Supplemental Fig. 6A). Recovery from this state

of heterologous tolerance requires longer periods than those that are produced by MOR-

-/- coupled NMDAR activity, and in σ1R mice, the restoration of analgesic effects could

not by accelerated σ1R antagonists (Fig. 8C).

The above observations indicate that the σ1R plays an essential role in bringing The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

MOR signaling under the negative control of NMDAR activity. Thus, we analyzed This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 15

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whether the deletion of σ1R influences the MOR-HINT1-NR1 association. Indeed, in

-/- σ1R mice, the MOR-NR1 association was greatly diminished, most likely because

HINT1 primarily associates with NR1 subunits (Fig. 9A). Thus, in the absence of σ1Rs

the MOR-NMDAR cross-regulation is impaired, and morphine produced almost no

recruitment of NMDAR activity, i.e., CaMKII autophosphorylation or weakening of the

residual MOR-NR1 association, molecular events that were not altered by the

administration of S1RA (Fig. 9B and Supplemental Fig. 6B). Interestingly, the in vivo

icv-injection of the recombinant σ1R restored the negative influence of NMDAR

-/- activation and diminished MOR-mediated morphine analgesia in σ1R mice, and this

effect was associated with a decrease in the binding of HINT1 proteins to NR1 subunits

(Fig. 9C).

The σ1R prevents the swapping of HINT1 proteins between MOR and NR1

subunits

Antioxidants & Redox Signaling The administration of S1RA 10 min or 30 min prior to morphine administration reduced

the opioid-induced phosphorylation of the NR1/NR2 subunits and promoted an early re-

association of NR1 subunits under MOR-HINT1 negative control, thereby limiting the

activity of the NMDAR-CaMKII pathway (Figs. 5C, 6A & 10A). However, longer

S1RA-morphine intervals, i.e., 1 h, 3 h and 24 h, brought about a delayed re-association

between the morphine-activated MORs and NR1 subunits (Fig. 10A). Interestingly, in

wild-type mice, morphine alone promoted certain transfer of HINT1 proteins from

MORs to NR1 subunits (Fig. 10A and Supplemental Fig. 7), and S1RA given before

morphine greatly increased this translocation. Thus, when S1RA was given from 1 h to

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) 24 h before morphine the transfer of HINT1 proteins increased. Under these

circumstances, PKC still acts on NR1 C1 S890/896/897 (Fig. 6A); however, the kinase This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 16

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is now with HINT1 at the NMDAR side and hardly reaches the MOR to promote those

changes that are responsible for acute analgesic tolerance. Thus, the MOR-NMDAR

-/- -/- physical and functional uncoupling was observed for HINT1 mice (48) and for σ1R

mice in this present study.

In the absence of morphine, σ1R antagonists disrupt σ1R-NR1 binding, which

facilitates the association of NR1 subunits with HINT1 (Fig. 7A). Notably, several

hours after S1RA administration and in the absence of morphine challenge, the basal

association between MORs and NR1s increased (Supplemental Fig. 8). It is under these

circumstances that morphine promotes the long-term transfer of HINT1 proteins to

NMDARs (Fig. 10A). Regarding the NR1-HINT1 interaction, in vitro PKC acts on NR1

C1 T879 and Ser 890, 896 (60), thereby abolishing HINT1 binding (Fig. 7A). However,

ex vivo data suggest that HINT1 binds to NR1 subunits in the presence of the serine

phosphorylation of C1 segment, such as S890 (Figs. 5C & 10A). Within the S1RA-

induced MOR-NMDAR complex and before morphine activates PKCγ, HINT1 could

bind to the NR1 region of T879. Thus, we evaluated whether HINT1 binds to PKCγ- Antioxidants & Redox Signaling phosphorylated T879A NR1 subunits, and the results indicate that this binding is indeed

possible (Fig. 10B). Interestingly, PKCγ increased the binding of NR1 subunits to σ1Rs,

indicating that in the absence of σ1R antagonists, the phosphorylation of C1 segment

could increase the ability of σ1Rs to remove MOR-HINT1 binding to NR1 subunits. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 17

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Discussion

The antinociceptive effects of opioids are related to their capacity to diminish calcium-

dependent neurotransmitter release. Thus, to prevent opioids from producing an

excessive reduction of neuronal excitability, NMDARs are recruited to the MOR

environment, where they become activated to restrain opioid signaling (45; 61). For this

control to be effective, the negative feedback must be proportional to the power of

MOR signaling. The disruption of this balance could provoke a disproportionate

NMDAR function, leading to cell damage, or to an excessive MOR activation, which

negatively affects cell homeostasis. The results of this study indicate that σ1Rs are

necessary to establish the NMDAR control on MOR signaling by facilitating the MOR-

HINT1-σ1R-NMDAR protein assembly. In this context, the σ1R cooperates with the

HINT1 protein to equilibrate the negative influence of NMDARs to the strength of the

MOR signals. This process is redox-regulated, and NOS inhibition or the targeted

deletion of either HINT1 or σ1R increases the MOR-mediated analgesic effects that

elude regulation through NMDAR activity (20; 48; 57; and present study). Interestingly,

Antioxidants & Redox Signaling σ1R antagonists affect MOR function comparable to the effect of the removal of

σ1R/HINT1 proteins, i.e., the impairment of the capacity of MORs to trigger NMDAR-

mediated restrain on opioid signaling. Notwithstanding, the experimental use of σ1R

antagonists in wild-type mice was successful in increasing MOR-mediated analgesic

effects without promoting tolerance. Therefore, this cellular mechanism can be

exploited without triggering an excessive MOR function, which could recruit alternative

systems to ultimately induce MOR hypofunction.

The σ1R as a ligand-regulated chaperone in its interaction with different proteins

could adopt different conformations. Thus, in its particular binding to NMDAR NR1 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) subunits at the cell membrane, the cellular and in vitro assays suggest that the σ1R loop This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 18

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faces the cytosolic side (4; 44; 55). The NR1 C terminal sequence is a target of the

calcium-binding protein CaM, which applies negative feedback to down-regulate the

gating of the NMDAR calcium channel in response to high levels of cytosolic Ca2+ (11).

The NR1 C1 amino acid sequence is essentially a positively charged domain that

includes the binding site for the σ1R loop that overlaps with that of the negatively

charged HINT1 protein (34) and with the Ca2+-CaM binding domain. The σ1R loop

binds specific regions on NR1 C0-C1 segments, while the σ1Rcd interacts with the NR1

C0 region. The σ1R has a SIM that could potentially bind a series of sumoylated

proteins in the MOR environment, such as the RGSZ2 (16). The relevance of these

SUMO-SIM regulatory interactions warrants further study.

In response to opioids, such as morphine, the σ1R is essential to bring the

NMDARs under the control of MOR-HINT1 complexes. This process is exquisitely

regulated, begins with the activation of MORs and requires the redox-regulated HINT1

protein, the redox zinc switch RGSZ2 protein and redox sensor proteins, such as PKC

and Raf-1 (15; 47; 50; 51; 54). In the absence of MOR activation, the HINT1 protein

Antioxidants & Redox Signaling carries the RGSZ2-nNOS assemblage that blocks MOR interaction with NMDARs.

Opioids release this barrier and enable the arrival of the NMDAR to the MOR-HINT1

complex through NO and the zinc-dependent binding of PKCγ to the HINT1 protein

(47; 50; 54). Within the MOR-NMDAR interaction, HINT1 binds the Ca2+-CaM site in

the NR1 C1 segment and inhibits NMDAR activity, whereas the σ1R covers the C0 and

the upper region of the C1 segment and reduces the overall affinity of NR1 subunits

towards HINT1 proteins (60). To build up the negative feedback on opioid signaling,

PKCγ phosphorylates the NR1 C1 Ca2+-CaM site to release the NMDAR from the

HINT1 inhibitory influence. The NMDAR is now ready to collaborate with the MOR to The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) increase PKCγ and CaMKII activities and make operative the control on opioid This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 19

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signaling (14). Whether the activity of the MORs reaches a certain threshold, PKCγ

increases the formation of reactive oxygen species (ROS) by acting on NOX/NADPH,

consolidating the long-term PKCγ activation that is required to regulate the Raf-

1/MAPK cascade and enhancing NMDAR function. Thus, NADPH/ROS production

and the sustained activation of PKCγ triggered by opioids are essential to develop and

maintain NMDAR-mediated tolerance to the analgesic effects of morphine (10; 23)

In the absence of HINT1, the MOR-NMDAR cross-regulation is disrupted, and

PKCγ cannot reach specific residues in the MOR C-terminal or third internal loop, as

required to desensitize responses to opioids and consequently, the capacity of morphine

-/- to produce antinociception increases (6; 9; 48). In σ1R mice, MORs and NMDARs

are also molecularly and functionally disconnected because the HINT1 protein swaps

MORs with NMDAR NR1 subunits. The cytosolic calcium levels regulate the

permeation of Ca2+ ions through the NMDAR pore; thus, σ1R binds to the NR1 subunit

in a calcium-dependent manner, blocking the entrance of Ca2+-CaM and also weakening

that of HINT1 proteins (55). In the presence of Ca2+-CaM, the removal of σ1Rs Antioxidants & Redox Signaling facilitates its inhibitory binding to the NR1 subunit to reduce NMDAR calcium fluxes.

However, reductions in the cytosolic calcium levels diminish the interaction of NR1

subunits with σ1Rs and the presence of Ca2+-CaM, thereby favoring NR1 binding to

HINT1 proteins. Under these circumstances, MOR-coupled HINT1 binds tightly to

Ca2+-CaM domains on NR1 subunits producing some inhibition of NMDAR function

(20; 63). Then, opioid-activated PKCγ binds to HINT1 and, acting on NR1 S890/896,

separates NR1-HINT1-PKC from the MOR. The transfer of the PKCγ scaffold, the

HINT1 protein, to NR1 subunits situates this kinase activity apart from MORs.

-/- The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) Accordingly, the in vivo administration of recombinant σ1Rs to σ1R mice removed This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 20

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the HINT1 binding from NR1 subunits and restored the negative influence of NMDARs

on MOR-mediated antinociception.

The role of σ1Rs in the function of at NMDAR suggests that σ1R ligands could

regulate the strength of MOR signaling. Indeed, it has been consistently reported that

σ1R agonists reduce and σ1R antagonists enhance morphine analgesia (25; 39). In in

vitro assays, agonists enhanced and antagonists reduced the association of σ1Rs with

NR1 subunits; however, the reducing effects of σ1R agonists on opioid analgesia are not

so evident. The release of endogenous σ1R agonists, most likely neurosteroids, could

mask the effect of the exogenous. The selective σ1R antagonist S1RA induces a 2-fold

reduction in the analgesic ED50s of different opioids against thermal stimuli (64; 70).

The effect of icv S1RA on morphine supraspinal analgesia depended on the interval

between the administration of these drugs, showing an optimal effect at approximately

10 min to 30 min and nearly no effect after 1 h. When S1RA was administered shortly

before morphine, the σ1R antagonist reduced the capacity of the opioid to activate the

NMDAR/CaMKII pathway in the MOR environment. Consequently, the negative

Antioxidants & Redox Signaling feedback of NMDARs on MOR signaling was impaired, and morphine analgesia

increased from the initial intervals post-opioid with almost no development of acute

tolerance.

Because the NMDAR contributes to the recruitment and activation of PKCγ in

the post-synapse (5), antagonists of σ1Rs when used at intervals that reduced the

morphine-induced activation of NMDARs also weakened that of PKCγ. The extension

of the S1RA-morphine interval for 3 h or 24 h did not potentiate morphine analgesia,

but some protection against acute tolerance was still observed. The molecular data

suggest that the administration of S1RA long before morphine treatment increases the The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) capacity of the opioid to swap the HINT1 proteins from MORs to NR1 subunits. In This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 21

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-/- σ1R mice, the HINT1 protein, to the detriment of its association with MORs, was

mostly found at NR1 subunits. This observation suggests that S1RA alters the

conformation of the σ1R (7; 33) and then removes its regulatory binding to the NR1

subunit. At this point, two scenarios could account for the influence of the S1RA-

morphine interval on morphine analgesia and NMDAR function: in the first scenario,

Ca2+ levels are provided by morphine-activated PLCβ, and S1RA, by disrupting σ1R-

NR1 binding, facilitates the access of Ca2+-CaM to reduce the activation that MOR-

PKCγ promotes on NMDARs. The reduction of NMDAR function diminishes the

cellular influx of Ca2+ and the local formation of Ca2+-CaM, and upon clearance of the

σ1R antagonist the σ1Rs bind again to NR1 subunits enabling in the absence of HINT1-

bound RGSZ2 the inhibitory re-association of NMDARs with MOR-HINT1 complexes.

Thus, S1RA removes the negative influence of NMDARs on MOR function and it

enhances morphine analgesia in naïve mice, as well as recovering the effects of

morphine in mice chronically treated with the opioid. The other scenario considers the

absence of MOR activation, low Ca2+ and, consequently, low Ca2+-CaM levels; in these Antioxidants & Redox Signaling circumstances S1RA, by removing the σ1R, promotes the tight binding of RGSZ2-free

MOR-HINT1 complexes to NR1 subunits. When morphine recruits PKCγ to the HINT1

protein, the absence of σ1Rs facilitates the swap of HINT1 from MORs to NR1

subunits. The activation of MORs increases, via PLCβ, calcium levels and that of Ca2+-

CaM, however PKCγ acting on NR1 S890 prevents Ca2+-CaM and HINT1 inhibitory

binding to CaM binding site on the NR1 subunit while not abrogating that of HINT1 to

the upstream region of the NR1 C1 segment. In these circumstances, PKCγ when bound

to HINT1-NR1 cannot transmit its negative influence on the MOR cytosolic residues,

and the morphine analgesic tolerance develops at a slower rate. The clearance of the The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

σ1R antagonist S1RA, together with the increases in local calcium, allows σ1R to This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 22

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disrupt NR1-HINT1 interaction and reestablish σ1R-NR1 and MOR-NR1 complexes.

Thus, the S1RA- and calcium-dependent status of the σ1R-NR1 association determines

whether HINT1 swaps partners between MOR and NR1 subunits.

Morphine alone also promoted the transfer of HINT1 proteins, but this effect

was limited in extent and reversible in the short term. In the absence of the σ1R

antagonist, the presence of calcium rapidly restored σ1R-NR1 binding and the

regulation of HINT1 through MORs. Thus, HINT1 swapping is under physiological

regulation by endogenous agonists of the σ1R (19). Notably, the neuroactive steroid

pregnenolone is released in response to drugs that act at GPCR-NMDAR complexes,

such as cannabinoids and opioids (62). Pregnenolone displays agonist activity at σ1Rs,

whereas its metabolite progesterone acts as an antagonist (37). However, pregnenolone

is rapidly converted into progesterone, and its effects are mostly associated with the

antagonism of σ1Rs (65). To delay this metabolism, pregnenolone is sulfated by

pregnenolone sulfotransferase (SULT2B1a), and this enzyme is induced in response to

events that are associated with NMDAR activity, such as AMPA receptors and NO (30).

Antioxidants & Redox Signaling Thus, σ1R agonists promote and antagonists reduce the activity of NMDARs,

and these effects have been documented in electrophysiological studies as changes in

NMDAR currents (36; 68) and variations in the PKC-mediated phosphorylation of NR1

subunits (26; 27). These profiles of neurosteroids on NMDAR activity when coupled

with the opioid-mediated activation of MOR most likely contribute to the regulation of

their analgesic effects. Thus, pregnenolone sulfate would promote NMDAR control of

MOR activity, and if the strength of MOR signaling produces an NMDAR activity that

compromises cell homeostasis, then pregnenolone could be converted into the σ1R

antagonist progesterone to uncouple NMDARs from the activating influence of MORs. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 23

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In addition, in neuropathic pain where NMDARs become over activated, the antagonists

of σ1Rs could disconnect the origin of such activation, and/or promote the binding of

negative regulators of NMDAR function, thereby demonstrating a therapeutic potential

(8; 52; 53; 70).

Innovation

In neural cells, the σ1R binds to NMDAR NR1 subunits and it co-operates with the

redox-regulated HINT1 protein to bring MOR signaling under the regulation of the

NMDAR, thereby contributing to tolerance. In this protein assembly opioids promote

the binding of the redox sensor PKCγ to the HINT1 histidine residues, via NO and zinc

metabolism, whereby the kinase recruits NMDAR activity proportional to MOR

signaling. In naïve mice, the σ1R antagonists disrupt σ1R-NR1 interaction and uncouple

the NMDAR from MOR activity, enhancing morphine analgesia and reducing the

development of acute opioid tolerance. In mice rendered tolerant to morphine, σ1R

antagonists promote the inhibition of NMDARs via Ca2+-CaM and they then increase

Antioxidants & Redox Signaling the strength of the MOR signaling, rescuing morphine analgesia from tolerance. Thus,

selective σ1R antagonists could be therapeutically exploited as adjuvants of opioid

analgesia, reducing the risk of adverse effects. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 24

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Materials and methods

In vitro interactions between recombinant proteins: pull-down of recombinant

proteins and phosphorylation assays.

A series of recombinant proteins were obtained (see Supplemental methods), and

having demonstrated that the σ1R, NR1 C0-C1-C2 or HINT1 proteins did no bind to

GST (100 nM: Genscript; USA Z02039) (55; 56), we determined the association of

σ1R with NMDAR NR1 subunits. The NR1 C-terminal sequence C0-C1-C2 or mutated

NR1 T879A were immobilized through covalent attachment to NHS-activated

sepharose 4 fast flow (#17-0906-01, GE, Spain) according to the manufacturer’s

instructions. The HINT1 protein (200 nM) and σ1R variants (100 nM) were incubated

either alone (negative control) or together with the immobilized proteins in 400 µL of a

buffer containing 50 mM Tris-HCl, pH 7.4 and 0.2% CHAPS and mixed by rotation for

30 min at RT. The influence of Ca2+ on this association was evaluated after the addition

of 2.5 mM CaCl2 to the media. After incubation, the pellets were obtained by

centrifugation, washed three times, solubilized in 2×Laemmli buffer and analyzed by

Antioxidants & Redox Signaling western blotting.

The regions involved in the interaction of NR1 C0-C1 with σ1R, HINT1 or

Calmodulin were investigated through peptide interference of binding using 13 peptides

(overlapping 5 residues) covering C0-C1 region of NR1. The interaction σ1R-NR1 was

also analyzed with peptides mapping regions in the σ1R loop and cdomain (Genscript;

USA). The purity of these peptides was higher than 95%.

PKCγ-mediated phosphorylation of the HINT1-RGSZ2 assembly. The Gα

subunits were previously incubated with 10 µM GTPγS in 50 µL of 10 mM HEPES, pH

7.4, 150 mM NaCl, 3 mM EDTA buffer (Gi2GTPγS) and the unbound GTPγS was The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) removed (centrifugal filter devices; 10 kDa nominal MW limit, Amicon Microcon YM- This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 25

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10#42407, Millipore). After 20 min, the HINT1 and RGSZ2 proteins were incorporated

into HINT1-RGSZ2 complexes, and then Gαi2GTPγS subunits were added to a final

100 nM and incubation continued for an additional period of 15 min. Subsequently, 30

in 100 µL of kinase buffer (60 mM HEPES-NaOH [pH 7.5]; 3 mM MgCl2; 3

mM MnCl2; 3 µM Na-orthovanadate; 1 mM DTT and 250 μM ATP) was added to the

mixture and the reaction was conducted at room temperature and it was terminated after

20 min by the addition of the PKC inhibitor Gö7874 (Calbiochem; #365252) at a

concentration of 5 µM.

The influence of SUMO1 on σ1R’s association with the NR1 C0-C1-C2

subunits was determined through the pre-incubation of recombinant σ1R (100 nM) with

agarose-SUMO1 for 30 min with rotation at room temperature in 150 µL of 50 mM

Tris-HCl, pH 7.5, 2.5 mM CaCl2 and 0.2% CHAPS. After the removal of free σ1R,

NR1 C0-C1-C2 (100 nM) was added to these proteins mixtures and incubated for an

additional 30 min. In a set of assays free SUMO1 was added to pre-formed agarose-

NR1-σ1R complexes (100 nM) for 30 min at RT. Agarose-SUMO1 pellets containing

Antioxidants & Redox Signaling the bound proteins were obtained by centrifugation, washed three times, solubilized in

2xLaemmli buffer and analyzed by western blotting.

Animals and evaluation of antinociception

-/- Wild-type and homozygous (σ1R ) male sigma receptor knockout mice, backcrossed

(N10 generation) onto a CD1 albino genetic background (Harlan Iberica, Spain), and

-/- homozygous (HINT1 ), generously supplied by I.B. Weinstein/J.B. Wang, were used

in this study (32; 51). The mice were housed and used in strict accordance with the

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) European Community guidelines for the Care and Use of Laboratory Animals (Council

Directive 86/609/EEC). All the procedures for handling and sacrificing the animals This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 26

Page 27 of 110

were approved by the Committee on Animal Care at CSIC. The animals were housed at

22º C under a 12 h light/dark cycle (lights on from 8 a.m. to 8 p.m.). Food and water

were provided ad libitum. The response of the animals to nociceptive stimuli was

assessed using the warm water (52 °C) tail-flick test. The tail-flick analgesic test applies

a thermal noxious stimulus to promote flicking of the mouse’s tail, and opioids given by

icv route increase the time elapsed between application of the stimulus and the flick.

This response comprises a spinal reflex which is under facilitator drive by the brain

stem nociceptive modulating network. After icv administration of opioids, the MORs in

the ventral region of PAG play an important role in the supraspinal pathways that

modulate spinal nociceptive processing (66; 67). Thus, icv morphine modulates

descending serotoninergic and adrenergic systems and inhibits responses to nociceptive

stimuli, including nociceptive withdrawal reflexes that are organized segmentally, such

as the hind limb withdrawal and tail flick reflexes (18). In this analgesic test, the

baseline latencies ranged from 1.7 to 2.0 seconds, and this parameter was not

significantly affected by the σ1R ligands or the solvent used ethanol/Cremophor

Antioxidants & Redox Signaling EL/physiological saline (1:1:18), 1.9 ± 0.2 seconds (n = 10). A cut-off time of 10

seconds was used to minimize the risk of tissue damage. Antinociception is expressed

as a percentage of the maximum possible effect (MPE = 100 x [test latency-baseline

latency]/[cut-off time-baseline latency]). Groups of 8 to 10 mice received a dose of

morphine, and antinociception was assessed at different time intervals thereafter.

Production of acute and chronic tolerance to the antinociceptive effect of

morphine.

The development of morphine-induced acute opioid tolerance was monitored as The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) described (15). Briefly, the animals received an icv priming dose of 10 nmol morphine This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 27

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in the right lateral ventricle. A 10 nmol dose produced 70-80% of the MPE in the “tail-

flick” test for analgesia. Controls were injected only with the opioid priming dose,

whereas the experimental groups received the drug under study prior to the morphine

priming dose or 24 h later few min (typically 30 min) before the morphine test dose of

10 nmol morphine. At this point, the analgesic effect of the 10 nmol priming dose had

dissipated as evidenced by the restoration of baseline latencies in the tail-flick test. In

some assays the desensitizing effect of icv administration of a priming dose of 10 nmol

morphine was addressed by an identical test dose of the opioid administered 24 and 48 h

later.

For the study of the time interval required to recover analgesic response from

acute tolerance the mice were all icv-injected with 10 nmol morphine and divided into

sub-groups of eight mice each. At increasing intervals post-opioid priming dose, a

different group was icv-injected with the morphine test dose of 10 nmol morphine. The

antinociceptive effect of morphine was evaluated at 30 min post-injection; allowing

time for the compound to reach its peak analgesic effect. Development of acute

Antioxidants & Redox Signaling tolerance was ascertained through the comparison of the effects promoted by the

morphine priming and test doses. Thus, data are expressed as the mean ± SEM from

groups of eight mice.

Mice were also pre-treated with a schedule of chronic morphine administration

(29). The mice were anesthetized by isoflurane inhalation prior to stereotaxically

implanting a sterile cannula in the lateral ventricle (coordinates: 0.3 mm caudal, 1 mm

lateral from bregma, and depth 2.3 mm) and fixing it to the skull with dental cement.

The animals were allowed to recover for 4 days before the experiments commenced and

icv injections of 10 nmol morphine per mouse were administered daily for 7 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) consecutive days. The placement of the cannula was verified for each mouse and only This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 28

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the data obtained from mice with a correctly inserted cannula were included in the

statistical analysis.

Immunohistochemistry

A rabbit IgG labeling kit (Zenon Tricolor Rabbit IgG labeling Kit #1; Molecular Probes,

Eugene, OR) was used for triple-labeling with the rabbit polyclonal IgGs against: σ1R

(internal region139-157, Invitrogen 42-3300; Alexa Fluor 488), NMDAR NR1 subunit

(C-terminus, Merk-Millipore, Chemicon AB9864; Alexa Fluor 555), and MOR (C-

terminal region, Abcam, ab 134054; Alexa Fluor 647). Mouse coronal brain sections

with the PAG were incubated with the labeling complex overnight and examined by

confocal microscopy (Leica TCS SP-5/LAS AF Lite Software, Microsystems, GmbH).

Controls for immunohistochemistry were performed according to standard protocols

(for further see details in Supplemental Methods and Supplemental Fig. 3).

Immunoprecipitation and western blotting

Antioxidants & Redox Signaling After icv morphine, groups of eight mice were killed at various intervals post-opioid;

PAG were obtained and processed to obtain the synaptosomal pellet as previously

described (49), and used for MOR and NR1 immunoprecipitation and co-precipitation

of HINT1, NR1 and MOR. This procedure has been described elsewhere (13; 17).

Further details in Supplemental Methods.

Statistical analysis

ANOVA, followed by the Student-Newman-Keuls test (SigmaStat, SPSS Science

Software, Erkrath, Germany) was performed, and significance was defined as p< 0.05. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 29

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Acknowledgements: We would like to thank Concha Bailón and Gabriela de Alba for

their excellent technical assistance. Cajal drawings were provided by the Legado Cajal,

Cajal Institute, CSIC, Madrid, Spain. This research was supported by the “Ministerio de

Sanidad y Consumo-Plan de Drogas 2011–2014” and the “Ministerio de Economía y

Competividad (MINECO), SAF 2012-34991”.

Author Disclosure Statement: The authors declare that, excluding income received

from our primary employer “Ministerio de Economía y Competitividad, MINECO” no

financial support or compensation has been received from any individual or corporate

entity over the past 3 years for research or professional services and that there are no

personal financial holdings that could be perceived as constituting a potential conflict of

interest. MM & JMV are full time employees at Esteve.

Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 30

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List of Abbreviations

Ca2+-CaM calcium calmodulin

CaMKII calcium and calmodulin dependent kinase II

CRD Cysteine-rich domain

CRM cholesterol-binding motif

ER endoplasmic reticulum

GPCR G protein coupled receptor

HINT1 histidine triad nucleotide binding protein 1

HR hydrophobic region

icv intracerebroventricular

MOR mu-opioid receptor

MPE maximum possible effect

NMDAR glutamate N-methyl-D-aspartate receptor

NO nitric oxide

nNOS neural nitric oxide synthase

NR1/2 subunit 1/2 of the glutamate NMDAR Antioxidants & Redox Signaling PAG periaqueductal grey

PKC protein kinase C

PMAR Potential Membrane Attachment Region

RGS regulators of G protein signaling

σ1R sigma 1 receptor

S1RA 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3- yl]oxy]ethyl] morpholine

SANR sumo-associated negative region

SBDL steroid-binding-like domain

SIM SUMO-interacting motif The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) SNAP (5)-Nitroso-N-acetylpenicillamine This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 31

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SUMO small ubiquitin-related modifier

TM transmembrane

TPEN N’,N’,N’,N’-tetrakis(2-pyridylmethyl) ethylenediamine Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 32

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Figure Legends

Figure 1.- The σ1R in the cell membrane: interaction with the glutamate NMDAR.

A, The protein domains of the murine σ1R and of the NMDAR NR1 cytosolic C

terminal sequence. The long isoform of σ1R contains 223 residues, with two

hydrophobic transmembrane domains, TM1 and TM2. The hairpin loop contains a

Sumo-Interacting Motif (SIM 61-65) within a hydrophobic region (HR) and a Sumo-

Associated Negative Region (SANR). The C-terminal domain comprises another

hydrophobic region (HR), which includes cholesterol-binding motifs (CRM1 and

CRM2), a Potential Membrane Attachment Region (PMAR) and Steroid-Binding-Like

Domain II (SBDLII). The NMDAR NR1 subunit C terminus C0-C1-C2 contains 104

residues with two hydrophobic regions (HR1 and HR2; Lasergene Protean Software

DNASTAR, Inc; Madison, WI, USA).

B, The σ1R cd region, charge average and hydrophobicity map (the images were

created using Lasergene Protean Software). The binding of this region to Bip is

regulated through calcium (* taken from 43), and the SBDLII associates with SBDLI in

Antioxidants & Redox Signaling the TM2 domain, forming the neurosteroid-binding pocket.

C, The binding of recombinant σ1R long and short forms to GST-NR1 C0-C1-C2. The

recombinant NR1 C-terminal sequence C0-C1-C2 and 1 receptor variants were used at

100 nM. The assay was performed in the presence or absence of 2.5 mM calcium. The

bait protein (GST-NR1 C0-C1-C2) was immobilized by covalent attachment to NHS-

activated sepharose. The prey proteins alone did not bind to either NHS-sepharose

(negative control) or recombinant GST (negative control). After incubation, the proteins

were resolved by SDS-PAGE chromatography, followed by western blotting analysis. P

stands for the precipitation of immobilized NR1 C-terminal sequences. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 46

Page 47 of 110

D, The proposed arrangement of the long σ1R in the cell membrane is shown with the

loop directed to the cytosol, the cd HR situated in the inner region of TM1 and TM2,

and the N- and C-terminal regions arranged towards the extracellular space.

(To see this illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars).

Figure 2.- Interference assay of the association between NR1 C0-C1 and σ1R, HINT1

and CaM.

A series of overlapping peptides (30 µM) mapping the C0 and C1 region (834-903) of

NR1 subunits were incubated with 100 nM σ1R, 200 nM HINT1 or 100 nM Ca2+-CaM

prior to the addition of 100 nM NR1. The NR1 was precipitated, and the associated

protein was evaluated through ECL-densitometry. These data were obtained from three

independent assays. *Significantly different from the σ1R or HINT1 signals in the

absence of interfering peptides, ANOVA-Student-Keuls test; p<0.05. The peptide

mapping regions that were tentatively implicated in the interaction of NR1 C0-C1 with

Antioxidants & Redox Signaling these proteins are indicated in bold. Ca2+-CaM basal binding was low and greatly

increased in the presence of peptides mapping to the HR1 and HR2 of NR1 subunits.

This binding was abrogated when NR1 was sequentially incubated with peptides 4 and

10 before adding Ca2+-CaM. To increase the readability, the frames indicate higher

exposition of the blots. The potential regions of Ca2+-CaM binding to the NR1 C0-C1

are indicated (Calmodulin Target Database;

http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html). (To see this illustration in

color the reader is referred to the web version of this article at

www.liebertonline.com/ars). The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 47

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Figure 3.- Juxtaposition of the σ1R and NR1 C0-C1 regions based on their charge

complementarities and hydrophobicity.

A, Proposed interaction of the σ1R loop with the NMDAR NR1 C0-C1 region. The

discontinuous frame indicates possible hydrophobic interactions. TM stands for

transmembrane region. The arrows on the NR1 sequence suggest a possible

intramolecular interaction between the HR1 and HR2 hydrophobic regions (Lasergene

Protean V8 DNASTAR).

B, Influence of SUMO1 on the σ1R association with NR1 subunits. The recombinant

1R protein (100 nM) was pre-incubated with agarose-SUMO1. After the removal of

the free σ1Rs, the NR1 subunits (100 nM) were added to the incubation milieu. In a set

of assays, pre-formed agarose-NR1-σ1R complexes (100 nM) were incubated with free

SUMO1. The agarose pellets containing the bound proteins were analyzed by western

blotting.

C, Model describing the potential interaction regions of the complete σ1R with NR1

C0-C1 segments. Closed boxes indicate charge complementarities, and open boxes Antioxidants & Redox Signaling indicate potential hydrophobic interaction. The arrows connect the hydrophobic region

on NR1 C0 with another region in σ1Rcd corresponding to SBDLII, a negative region

that could bind calcium. TM stands for transmembrane domain, and the helix

organization (H2-H5) is taken from (43).

D, Diagram of calcium-dependent σ1R binding to the NR1 C terminal C0-C1 region.

The σ1R loop in the cell membrane is oriented toward the cytosolic side, and both the N

and C terminal sequences face the extracellular space. The hydrophobic cd region is

oriented toward the inner side of the membrane and between the TM domains, forming

the ligand-binding pocket. This hydrophobic and negative cd region interacts with a The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) positive region of the σ1R loop. The position of certain residues is indicated in the This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 48

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figure. Upon calcium binding, this negative cd region becomes neutral and subsequently

releases the positive loop region to bind other proteins, such as the NR1 subunit.

Peptide interference mapping suggests that the NR1 HR1 and HR2 hydrophobic regions

establish an intramolecular interaction. The calcium-bound σ1Rcd region would disrupt

this internal interaction to bind to the NR1 subunit.

Figure 4.- Co-localization of the NMDAR NR1 subunit with σ1R and MOR in the mouse

PAG

Upper panel, Original Cajal drawing showing cells of the PAG, Golgi method. A:

cerebral aqueduct; cell shape is varied, with predominance of the fusiform type, and the

orientation is oblique or transversal. Coronal drawing of mouse brain showing the PAG

region analyzed in this triple co-localization of σ1R, NMDAR NR1 subunit and MOR

(red square in the diagram). Middle panel, Confocal laser-scanning microphotographs

taken from coronal histological sections (10 µm) through the midbrain PAG showing

individual labeling for σ1R (green), NR1 (red), and MOR (blue) antigens.

Antioxidants & Redox Signaling Immunoreactivity was visualized with Alexa Fluor 488, 555 and 647, respectively.

Lower panel, Triple co-localization of the MOR, σ1R and NR1 subunits could be

observed at the cell periphery and fibers (nuclei were removed). There was a high

degree of coincidence between NR1 and σ1R (yellow color). The MOR co-localizes

with σ1R (light green color) and NR1 (purple color) in a discrete pattern. Notice that

high-power magnification panels show triple-co-localization as white structures (red

arrows, for details see Methods and Supplemental Fig. 3).

Figure 5.- Effect of icv σ1R ligands on the morphine supraspinal analgesia and NMDAR The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) activity This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 49

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A, Influence of the interval between the selective antagonist of σ1R, S1RA, and

morphine in the production of analgesia. Mice received icv 3 nmol S1RA at the

indicated time intervals before 3 nmol morphine, and analgesia was evaluated in the

thermal (water 52 °C) “tail-flick” test 30 min later. The bars represent the mean ± SEM

of the data from 6 mice. Significantly different from the group that received only

morphine, p<0.05.

A series of σ1R antagonists and the agonist PRE084 were icv-injected at 30 min before

3 nmol morphine, and antinociception was evaluated at the indicated post-opioid

intervals. Each point represents the mean ± SEM of data from 10 mice. The area under

the curve in the post-morphine interval 15 min to 90 min was calculated using the

trapezoidal rule for each σ1R ligand and dose (Sigmaplot/Sigmastat v12.5, Erkrath,

Germany). The analgesia produced by the σ1R antagonist-morphine combination was

significantly different from that of morphine alone, ANOVA-Student-Newman-Keuls,

p<0.05. The σ1R agonist PRE084 prevented S1RA from enhancing morphine analgesia.

Mice received a combined injection of 3 nmol PRE084 and 3 nmol S1RA 30 min before Antioxidants & Redox Signaling morphine treatment. Significantly different from the group that received vehicle and

morphine or PRE084+S1RA and morphine, p<0.05.

B, The neurosteroids progesterone (PG), pregnenolone (PN), PN acetate and PN sulfate

(sulf) were all used at 3 nmol, icv. The enhancing effects of progesterone on morphine

analgesia were abolished by pregnenolone sulfate.  Significantly different from the

group that received morphine and vehicle instead of the neurosteroid, p<0.05.

C, The selective σ1R antagonist S1RA impairs the MOR-mediated activation of

NMDARs. Groups of 42 mice each received an icv dose of 10 nmol morphine alone or

3 nmol S1RA 30 min before the opioid. Control mice were treated with saline instead of The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

morphine. Subsequently, for each group, 6 mice were sacrificed at the indicated This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 50

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intervals. PAG synaptosomes were obtained to determine ex vivo the presence of

CaMKII P-Thr286, NR1 C1 P-Ser890, and NR2A P-Tyr 1325. The MOR proteins were

immunoprecipitated, and the associated NR1 subunits were determined by western

blotting. Immunosignals (average optical density of the pixels within the object

area/mm2; Quantity One Software, BioRad) were expressed as the change relative to the

control group (attributed an arbitrary value of 1, dashed lines). Each bar represents the

mean ± SEM of the data from three determinations that were performed using different

gels and blots. For every post-opioid interval, indicates that S1RA produced a

significant difference from the group receiving only morphine, p<0.05. We observed no

differences in the levels of the non-phosphorylated proteins. Representative blots are

shown in Supplemental Fig. 4.

The diagram indicates that S1RA impairs the MOR to NMDAR pathway, which is

required to build up the negative feedback via kinases such as CaMKII on opioid

signaling.

Antioxidants & Redox Signaling Figure 6.- Influence of the interval S1RA-morphine on the MOR-induced activation of

NMDARs.

A, Influence of the interval S1RA-morphine. Groups of 36 mice each received icv a

desensitizing dose of 10 nmol morphine alone or 3 nmol S1RA at 10 min, 30 min, 1 h, 3

h or 24 h before the administration of the opioid. Subsequently, for each group

receiving morphine, 6 mice were sacrificed at 90 min, 6 h or 24 h after opioid treatment.

The control mice received saline instead of morphine. PAG synaptosomes were

obtained to determine the presence of CaMKII P-Thr286, NR1 C1 P-Ser890 and P-

Ser897, NR2A P-Tyr 1325. The assay was repeated at least twice. The data from the The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) S1RA-morphine intervals that failed to enhance analgesia but conferred protection from This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 51

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tolerance are framed. For every post-opioid interval, the symbol indicates that S1RA

produced a significant difference from the group receiving only morphine, ANOVA-

Student-Newman-Keuls test, p<0.05. The details are shown in Fig. 5.

B, Protection against morphine acute antinociceptive tolerance. A priming icv dose of

10 nmol morphine reduced the analgesic response to a second and identical test dose of

the opioid when administered 24 h later. Increasing doses of S1RA, BD1047 and

BD1063 were icv-injected at 30 min before the morphine priming dose, and the

analgesic effect of the test dose was evaluated 24 h later. Analgesia was measured in the

thermal tail-flick test at the peak effect for morphine analgesia, 30 min post-opioid

treatment. Effect of the interval between S1RA and the morphine priming dose on acute

tolerance. The σ1R antagonist S1RA was administered icv at 10 min, 30 min, 1 h or 24

h before the morphine priming dose (10 nmol), and the analgesic effects of identical

doses were evaluated 24 h (test dose 1) and 48 h (test dose 2) later. Each bar indicates

the mean ± SEM of the analgesia; n=6 mice. Significantly different from the group that

received saline instead of the σ1R ligand before morphine priming dose, p<0.05. Antioxidants & Redox Signaling C, Mice were administered morphine daily for 6 days (10 nmol, icv) and antinociception

was evaluated by the tail flick test 30 min after injection. On day 7, the administration

of S1RA (3 nmol) to morphine tolerant mice restored the antinociceptive effect of the

opioid. Each point/bar represents the mean ± SEM of the data from 10 mice. *

Significantly different from the group that received the first dose of morphine (day 0),

p<0.05. Immunodetection of NMDAR-related signals in the PAG of mice not exposed

to morphine (control), mice that received morphine for 7 days and mice that after 6 days

of morphine treatment received 3 nmol S1RA plus 10 nmol morphine on day 7. *

Significantly different from the control group that received saline instead of morphine, The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) p<0.05. Details as in Fig. 5C. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 52

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Figure 7.- The redox-regulated coupling of NMDARs with MORs: effect of σ1R ligands.

A, MOR-mediated activation of NMDARs: І) Zinc-mediated association of PKCγ with

the HINT1 protein. Zinc was removed from the recombinant proteins (TPEN-EDTA

buffer) prior incubation of 200 nM HINT1 with 100 nM GST-PKC in presence of

ZnCl2. GST alone did not bind to the HINT1 protein. CRD stands for cysteine-rich

domain and P for precipitation of the GST protein with glutathione-sepharose (GS). A

similar study was conducted between 100 nM GST-RGSZ2 and HINT1. ІІ) The

RGSZ2, PKC and HINT1 form a ternary complex: 200 nM HINT1 were incubated

with 100 nM GST-RGSZ2 in the presence of 100 nM ZnCl2 and increasing

concentrations of PKCγ. Details as in (І). ІІІ) PKC disrupts HINT1-RGSZ2 association

in presence of GGTPγS subunits. GST-RGSZ2 (100 nM) and HINT1 (200 nM) were

incubated in the absence or presence of 100 nM Gαi2GTPγS and of PKC. ІV) Effect of

PKC on RGSZ2 and HINT1. RGSZ2 (100 nM) or HINT1 (100 nM) were incubated for

20 min at RT with 30 nM PKCγ. The PKCγ-induced phosphorylation of RGSZ2 and Antioxidants & Redox Signaling HINT1 was evaluated using specific anti-phospho antibodies. V) Effect of HINT1

phosphorylation on its association with RGSZ2 and the C-terminal cytosolic region of

NR1 subunit (C0-C1-C2). Native and PKC-phosphorylated HINT1 proteins (200 nM)

were incubated with either 100 nM GST-RGSZ2 or GST-NR1. VІ) Phosphorylation of

NR1 C0-C1-C2 by PKC, and (VІІ) its association with HINT1 and σ1R. Further details

in Methods.

B, Left: Effect of progesterone (PG) and pregnenolone (PN) sulfate on the association

σ1R-NR1 C0-C1-C2. Agarose-NR1 was pre-incubated in the presence of 2.5 mM CaCl2

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) (30 min, RT) with the σ1R (100 nM) before the addition of 30 µM PG or PN (30 min,

RT). P indicates that agarose was recovered and washed before the analysis of the NR1- This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 53

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bound σ1R through SDS-PAGE and western blotting (WB). Each bar represents the

mean ± SEM of three determinations using different gels and blots. Significant

differences with respect the control without neurosteroid, ANOVA-Student-Newman-

Keuls test; p<0.05. Right: Influence of increasing concentrations of PG on the

association of NR1 with σ1Rs. Agarose-NR1 was incubated with the σ1R before the

addition of PG. Agarose-NR1 carrying the associated σ1Rs was recovered and washed

before the addition of 200 nM HINT1 or 100 nM CaM (in the presence of 2.5 mM

CaCl2 and of 30 µM peptide 4). Low: This assay was also conducted with σ1R

antagonists (S1RA and BD1047) and the agonist PRE084 in the absence or presence of

CaM.

Diagram: S1RA removes the σ1R from the NR1 subunit, favoring the binding of Ca2+-

CaM, which impairs the interaction of NMDARs with MOR-HINT1 complexes. As a

result, morphine only recruits a fraction of the NMDAR activity that is required to

control its effects. (To see this illustration in color the reader is referred to the web

version of this article at www.liebertonline.com/ars). Antioxidants & Redox Signaling

Figure 8.- The MOR-NMDAR cross-regulation requires NO and zinc metabolism: the

role of σ1Rs.

-/- A, Because σ1R mice display enhanced antinociception to opioids, the effect of the

direct activation of NMDARs by icv-injection of NMDA and of NOS inhibition, 3 nmol

morphine was studied instead of 10 nmol used in wild-type mice. Saline or 50 pmol

NMDA, 7 nmol L-NNA were icv-injected at 20 min before morphine treatment into

-/- wild-type and σ1R mice, and analgesia was determined using in the warm water tail-

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) flick test. Significantly different from the group that received saline and morphine,

ANOVA-Student-Newman-Keuls test, p<0.05. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 54

Page 55 of 110

B, NOS provides the zinc ions that are required for the recruitment of PKCγ to the

HINT1 protein in the MOR environment. PAG synaptosomes obtained from wild-type

-/- -/- mice with and without in vivo inhibition of NOS, σ1R mice, and HINT1 mice were

incubated for 4 h at 4°C with ZnCl2, the NO generator SNAP, and the metal ion chelator

TPEN. Subsequently, free zinc ions were removed by centrifugation and extensive

washing. The synaptosomal membranes were then solubilized and incubated with

affinity-purified IgGs raised against extracellular sequences in MOR. The MOR-

associated proteins were then separated by SDS-PAGE and analyzed by western

blotting. Doses of reactives and the incubation time were taken from (51; 54). IP

signifies immunoprecipitation and WB western blot analysis.

C, Rescue of morphine acute analgesic tolerance via inhibition of NMDAR, NOS or

PKC. A morphine priming dose of 10 nmol was icv-injected, and analgesia evaluated 30

min later in the “tail-flick” test. The non-competitive NMDAR antagonist MK801 (1

nmol), the NOS inhibitors L-NNA (7 nmol) and L-NAME (20 nmol), or the PKC

inhibitor Gö7874 (1 nmol), were icv-injected 30 min before administering 24 h later an Antioxidants & Redox Signaling identical morphine test dose of 10 nmol, and analgesia was again measured 30 min

later. Each bar indicates the mean ± SEM of the analgesia; n=8 mice. Significantly

different from the group that received saline before the morphine test dose, p<0.05.

Recovery from morphine acute antinociceptive tolerance produced by a single dose of

-/- 10 nmol morphine in wild-type and σ1R mice: effect of the antagonist of σ1Rs,

S1RA. After icv-injecting all of the mice with the morphine priming dose of 10 nmol,

the analgesia was evaluated in the thermal tail-flick 30 min later. At the time intervals

that are indicated in the figure a different group of 4 mice received a test dose of 10

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) nmol morphine, and analgesia was subsequently evaluated 30 min later. *Significantly

different from the control group that received the priming dose of morphine, ANOVA, This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 55

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Student-Newman-Keuls test, p<0.05. In a parallel assay, the mice received 1 nmol

S1RA 30 min before the morphine priming dose. The evaluation of antinociceptive

tolerance was as described above. *Significantly different from the control group that

received S1RA and the priming dose of morphine, p<0.05.

Figure 9.- The σ1R connects MOR with the negative regulation of NMDAR

A, Presence of NR1 subunits, HINT1 and σ1R in the synaptosomes of PAG obtained

-/- from wild-type and σ1R mice. IP: MOR or NR1 was immunoprecipitated, and the co-

precipitated proteins were detected by western blotting (WB). Significantly different

from the paired group (n = 3), ANOVA, Student-Newman-Keuls test, p<0.05. WT

-/- (wild-type mice), KO (σ1R mice) and P2 (synaptosomal fraction).

-/- B, Morphine promotes little activation of the NMDAR-CaMKII pathway in σ1R

mice. Mice were icv-injected with 10 nmol morphine, and ex vivo determinations were

-/- performed at the indicated post-opioid time intervals. In σ1R mice, MOR binding to Antioxidants & Redox Signaling NR1 subunits was impaired, and then morphine hardly altered the association that

remained. Moreover, in the absence of σ1Rs, morphine recruited little CaMKII activity

and only for a short interval. Details in Fig. 5C and Supplemental Fig. 4.

-/- C, The icv-injection of the recombinant σ1R in σ1R mice reduced the antinociceptive

potency of morphine. The mice were icv-injected with 0.5 nmol recombinant σ1R, and

the effect of morphine was evaluated 24 h later. Significantly different from the group

that received saline instead of the σ1R, p<0.05. The icv-injection of the σ1R restored

-/- the NMDAR negative regulation on MORs. The σ1R mice that had received the σ1R The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) were icv-injected with 50 pmol NMDA and 10 nmol morphine 24 h later. Significantly This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 56

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different from the group that received saline instead of NMDA, p<0.05. Inset:

-/- Solubilized brain membranes from σ1R mice were incubated with biotinylated IgGs

directed against the NR1. After recovery with streptavidin-sepharose (P), the NR1-

containing complexes were exposed to σ1R recombinant protein (200 nM) before

detecting HINT1-associated proteins by SDS-PAGE chromatography and western-

blotting (WB). This study was repeated at least twice using different preparations. Equal

loading was determined from the NR1 signal.

Figure 10.- The σ1R antagonist S1RA transfers HINT1 proteins from morphine-

activated MORs to NMDAR NR1 subunits.

A, Groups of 48 mice each received an icv injection of only morphine or 3 nmol S1RA

10 min, 30 min, 1 h, 3 h or 24 h before treatment with 10 nmol morphine. A total of 8

mice from each group were sacrificed 90 min, 6 h or 24 h after opioid treatment.

Control mice received saline instead of S1RA or morphine. The MORs were

immunoprecipitated (IP) from PAG synaptosomes, and the co-precipitated NR1 Antioxidants & Redox Signaling subunits were immunodetected by western blotting (WB). Significantly different from

the respective control group that received saline instead of morphine, p<0.05.

Association of HINT1 with MORs and NR1 subunits. In the experimental groups

described above, the association of HINT1 proteins with MORs and NR1 subunits was

determined for control, morphine- and S1RA-morphine-treated groups 90 min and 24 h

post-morphine treatment. The circle shows that morphine alone induces the transfer of

HINT1 to NR1 subunits. The S1RA-morphine intervals that did not enhance analgesia

but conferred protection from tolerance are framed. Controls of MOR and NR1

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) immunoprecipitation are shown in Supplemental Fig. 7. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 57

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The diagrams represent the framed S1RA-morphine intervals. S1RA administered 1 h

or 24 before morphine did not enhance analgesia or alter the MOR-promoted NMDAR

activity at 24 h, as indicated by NMDAR () (Fig. 5A & 6A). However, some

protection against analgesic tolerance was observed (Fig. 6B). Upon the S1RA-

mediated removal of σ1Rs, the NR1 subunit binds tightly to HINT1 at the MOR C

terminus. HINT1 carries the activated PKCγ that acts on NR1 C1 S890/896, promoting

the separation of the MOR C terminus from NR1-HINT1. Under these circumstances,

CaMKII and PKC barely reach the MOR, and then analgesic tolerance to morphine

develops at a slower rate (6; 9; 48).

B, Native or mutated NR1 T879A (100 nM) binds similarly to HINT1 (200 nM). While

PKC-phosphorylated native NR1 displayed poor affinity for HINT1, PKC-

phosphorylated NR1 T879A bound to the HINT1 protein. The phosphorylation of either

form of NR1 greatly augmented its association with σ1Rs. (To see this illustration in

color the reader is referred to the web version of this article at

www.liebertonline.com/ars). Antioxidants & Redox Signaling

Page 59 of 110

59 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 59

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Figure 1.- The σ1R in the cell membrane: interaction with the glutamate NMDAR.

A, The protein domains of the murine σ1R and of the NMDAR NR1 cytosolic C

terminal sequence. The long isoform of σ1R contains 223 residues, with two

hydrophobic transmembrane domains, TM1 and TM2. The hairpin loop contains

a Sumo-Interacting Motif (SIM 61-65) within a hydrophobic region (HR) and a

Sumo-Associated Negative Region (SANR). The C-terminal domain comprises

another hydrophobic region (HR), which includes cholesterol-binding motifs

(CRM1 and CRM2), a Potential Membrane Attachment Region (PMAR) and

Steroid-Binding-Like Domain II (SBDLII). The NMDAR NR1 subunit C terminus

C0-C1-C2 contains 104 residues with two hydrophobic regions (HR1 and HR2;

Lasergene Protean Software DNASTAR, Inc; Madison, WI, USA).

B, The σ1R cd region, charge average and hydrophobicity map (the images were

created using Lasergene Protean Software). The binding of this region to Bip is

regulated through calcium (* taken from 43), and the SBDLII associates with

SBDLI in the TM2 domain, forming the neurosteroid-binding pocket.

Antioxidants & Redox Signaling C, The binding of recombinant σ1R long and short forms to GST-NR1 C0-C1-C2.

The recombinant NR1 C-terminal sequence C0-C1-C2 and σ1 receptor variants

were used at 100 nM. The assay was performed in the presence or absence of 2.5

mM calcium. The bait protein (GST-NR1 C0-C1-C2) was immobilized by covalent

attachment to NHS-activated sepharose. The prey proteins alone did not bind to

either NHS-sepharose (negative control) or recombinant GST (negative control).

After incubation, the proteins were resolved by SDS-PAGE chromatography,

followed by western blotting analysis. P stands for the precipitation of immobilized

NR1 C-terminal sequences. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 60

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D, The proposed arrangement of the long σ1R in the cell membrane is shown with

the loop directed to the cytosol, the cd HR situated in the inner region of TM1 and

TM2, and the N- and C-terminal regions arranged towards the extracellular space.

(To see this illustration in color the reader is referred to the web version of this

article at www.liebertonline.com/ars).

σ Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 61

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62 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 62

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Figure 2.- Interference assay of the association between NR1 C0-C1 and σ1R,

HINT1 and CaM.

A series of overlapping peptides (30 µM) mapping the C0 and C1 region (834-903)

of NR1 subunits were incubated with 100 nM σ1R, 200 nM HINT1 or 100 nM

Ca2+-CaM prior to the addition of 100 nM NR1. The NR1 was precipitated, and

the associated protein was evaluated through ECL-densitometry. These data were

obtained from three independent assays. *Significantly different from the σ1R or

HINT1 signals in the absence of interfering peptides, ANOVA-Student-Keuls test;

p<0.05. The peptide mapping regions that were tentatively implicated in the

interaction of NR1 C0-C1 with these proteins are indicated in bold. Ca2+-CaM

basal binding was low and greatly increased in the presence of peptides mapping to

the HR1 and HR2 of NR1 subunits. This binding was abrogated when NR1 was

sequentially incubated with peptides 4 and 10 before adding Ca2+-CaM. To

increase the readability, the frames indicate higher exposition of the blots. The

potential regions of Ca2+-CaM binding to the NR1 C0-C1 are indicated

Antioxidants & Redox Signaling (Calmodulin Target Database;

http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html). (To see this

illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars). The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 63

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64 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 64

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Figure 3.- Juxtaposition of the σ1R and NR1 C0-C1 regions based on their charge

complementarities and hydrophobicity.

A, Proposed interaction of the σ1R loop with the NMDAR NR1 C0-C1 region. The

discontinuous frame indicates possible hydrophobic interactions. TM stands for

transmembrane region. The arrows on the NR1 sequence suggest a possible

intramolecular interaction between the HR1 and HR2 hydrophobic regions

(Lasergene Protean V8 DNASTAR).

B, Influence of SUMO1 on the σ1R association with NR1 subunits. The

recombinant σ1R protein (100 nM) was pre-incubated with agarose-SUMO1. After

the removal of the free σ1Rs, the NR1 subunits (100 nM) were added to the

incubation milieu. In a set of assays, pre-formed agarose-NR1-σ1R complexes (100

nM) were incubated with free SUMO1. The agarose pellets containing the bound

proteins were analyzed by western blotting.

C, Model describing the potential interaction regions of the complete σ1R with

NR1 C0-C1 segments. Closed boxes indicate charge complementarities, and open

Antioxidants & Redox Signaling boxes indicate potential hydrophobic interaction. The arrows connect the

hydrophobic region on NR1 C0 with another region in σ1Rcd corresponding to

SBDLII, a negative region that could bind calcium. TM stands for transmembrane

domain, and the helix organization (H2-H5) is taken from (43).

D, Diagram of calcium-dependent σ1R binding to the NR1 C terminal C0-C1

region.

The σ1R loop in the cell membrane is oriented toward the cytosolic side, and both

the N and C terminal sequences face the extracellular space. The hydrophobic cd

region is oriented toward the inner side of the membrane and between the TM The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) domains, forming the ligand-binding pocket. This hydrophobic and negative cd This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 65

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region interacts with a positive region of the σ1R loop. The position of certain

residues is indicated in the figure. Upon calcium binding, this negative cd region

becomes neutral and subsequently releases the positive loop region to bind other

proteins, such as the NR1 subunit.

Peptide interference mapping suggests that the NR1 HR1 and HR2 hydrophobic

regions establish an intramolecular interaction. The calcium-bound σ1Rcd region

would disrupt this internal interaction to bind to the NR1 subunit.

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67 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 67

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Figure 4.- Co-localization of the NMDAR NR1 subunit with σ1R and MOR in the

mouse PAG

Upper panel, Original Cajal drawing showing cells of the PAG, Golgi method. A:

cerebral aqueduct; cell shape is varied, with predominance of the fusiform type,

and the orientation is oblique or transversal. Coronal drawing of mouse brain

showing the PAG region analyzed in this triple co-localization of σ1R, NMDAR

NR1 subunit and MOR (red square in the diagram). Middle panel, Confocal laser-

scanning microphotographs taken from coronal histological sections (10 µm)

through the midbrain PAG showing individual labeling for σ1R (green), NR1

(red), and MOR (blue) antigens. Immunoreactivity was visualized with Alexa

Fluor 488, 555 and 647, respectively. Lower panel, Triple co-localization of the

MOR, σ1R and NR1 subunits could be observed at the cell periphery and fibers

(nuclei were removed). There was a high degree of coincidence between NR1 and

σ1R (yellow color). The MOR co-localizes with σ1R (light green color) and NR1

(purple color) in a discrete pattern. Notice that high-power magnification panels

Antioxidants & Redox Signaling show triple-co-localization as white structures (red arrows, for details see Methods

and Supplemental Fig. 3). The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 68

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69 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 69

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Figure 5.- Effect of icv σ1R ligands on the morphine supraspinal analgesia and

NMDAR activity

A, Influence of the interval between the selective antagonist of σ1R, S1RA, and

morphine. Mice received icv 3 nmol S1RA at the indicated time intervals before 3

nmol morphine, analgesia was evaluated 30 min later. The bars represent the

mean ± SEM of the data from 6 mice. *Significantly different from the group that

received only morphine, p<0.05.

A series of σ1R antagonists and the agonist PRE084 were icv-injected at 30 min

before 3 nmol morphine, and antinociception was evaluated at the indicated post-

opioid intervals. Each point represents the mean ± SEM of data from 10 mice. The

area under the curve in the post-morphine interval 15 min to 90 min was

calculated using the trapezoidal rule for each σ1R ligand and dose

(Sigmaplot/Sigmastat v12.5, Erkrath, Germany). *The analgesia produced by the

σ1R antagonist-morphine combination was significantly different from that of

morphine alone, ANOVA-Student-Newman-Keuls, p<0.05. The σ1R agonist

Antioxidants & Redox Signaling PRE084 prevented S1RA from enhancing morphine analgesia. Mice received a

combined injection of 3 nmol PRE084 and 3 nmol S1RA 30 min before morphine

morphine or PRE084+S1RA and morphine, p<0.05.

B, The neurosteroids progesterone (PG), pregnenolone (PN), PN acetate and PN

sulfate (sulf) were all used at 3 nmol, icv. The enhancing effects of progesterone on

morphine analgesia were abolished by pregnenolone sulfate. *Significantly

different from the group that received morphine and vehicle instead of the

neurosteroid, p<0.05. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 70

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C, The selective σ1R antagonist S1RA impairs the MOR-mediated activation of

NMDARs. Groups of 42 mice each received an icv dose of 10 nmol morphine alone

or 3 nmol S1RA 30 min before the opioid. Control mice were treated with saline

instead of morphine. Subsequently, for each group, 6 mice were sacrificed at the

indicated intervals. PAG synaptosomes were obtained to determine ex vivo the

presence of CaMKII P-Thr286, NR1 C1 P-Ser890, and NR2A P-Tyr 1325. The

MOR proteins were immunoprecipitated, and the associated NR1 subunits were

determined by western blotting. Immunosignals (average optical density of the

pixels within the object area/mm2; Quantity One Software, BioRad) were

expressed as the change relative to the control group (attributed an arbitrary value

of 1, dashed lines). Each bar represents the mean ± SEM of the data from three

determinations that were performed using different g

post-opioid interval, indicates that S1RA produced a significant difference from

the group receiving only morphine, p<0.05. We observed no differences in the

levels of the non-phosphorylated proteins. Representative blots are shown in

Antioxidants & Redox Signaling Supplemental Fig. 4.

The diagram indicates that S1RA impairs the MOR to NMDAR pathway, which is

required to build up the negative feedback via kinases such as CaMKII on opioid

signaling. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 71

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72 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 72

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Figure 6.- Influence of the interval S1RA-morphine on the MOR-induced

activation of NMDARs.

A, Influence of the interval S1RA-morphine. Groups of 36 mice each received icv a

desensitizing dose of 10 nmol morphine alone or 3 nmol S1RA at 10 min, 30 min, 1

h, 3 h or 24 h before the administration of the opioid. Subsequently, for each group

receiving morphine, 6 mice were sacrificed at 90 min, 6 h or 24 h after opioid

treatment. The control mice received saline instead of morphine. PAG

synaptosomes were obtained to determine the presence of CaMKII P-Thr286, NR1

C1 P-Ser890 and P-Ser897, NR2A P-Tyr 1325. The assay was repeated at least

twice. The data from the S1RA-morphine intervals that failed to enhance analgesia

but conferred protection from tolerance are framed. *For every post-opioid

interval, the symbol indicates that S1RA produced a significant difference from

the group receiving only morphine, ANOVA-Student-Newman-Keuls test, p<0.05.

The details are shown in Fig. 5.

B, Protection against morphine acute antinociceptive tolerance. A priming icv dose

Antioxidants & Redox Signaling of 10 nmol morphine reduced the analgesic response to a second and identical test

dose of the opioid when administered 24 h later. Increasing doses of S1RA, BD1047

and BD1063 were icv-injected at 30 min before the morphine priming dose, and

the analgesic effect of the test dose was evaluated 24 h later. Analgesia was

measured in the thermal tail-flick test at the peak effect for morphine analgesia, 30

min post-opioid treatment. Effect of the interval between S1RA and the morphine

priming dose on acute tolerance. The σ1R antagonist S1RA was administered icv

at 10 min, 30 min, 1 h or 24 h before the morphine priming dose (10 nmol), and the

analgesic effects of identical doses were evaluated 24 h (test dose 1) and 48 h (test The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) dose 2) later. Each bar indicates the mean ± SEM of the analgesia; n=6 mice. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 73

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ificantly different from the group that received saline instead of the σ1R

ligand before morphine priming dose, p<0.05.

C, Mice were administered morphine daily for 6 days (10 nmol, icv) and

antinociception was evaluated by the tail flick test 30 min after injection. On day 7,

the administration of S1RA (3 nmol) to morphine tolerant mice restored the

antinociceptive effect of the opioid. Each point/bar represents the mean ± SEM of

the data from 10 mice. * Significantly different from the group that received the

first dose of morphine (day 0), p<0.05. Immunodetection of NMDAR-related

signals in the PAG of mice not exposed to morphine (control), mice that received

morphine for 7 days and mice that after 6 days of morphine treatment received 3

nmol S1RA plus 10 nmol morphine on day 7. * Significantly different from the

control group that received saline instead of morphine, p<0.05. Details as in Fig.

5C. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 74

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75 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 75

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Figure 7.- The redox-regulated coupling of NMDARs with MORs: effect of σ1R

ligands.

A, MOR-mediated activation of NMDARs: І) Zinc-mediated association of PKCγ

with the HINT1 protein. Zinc was removed from the recombinant proteins (TPEN-

EDTA buffer) prior incubation of 200 nM HINT1 with 100 nM GST-PKCγ in

presence of ZnCl2. GST alone did not bind to the HINT1 protein. CRD stands for

cysteine-rich domain and P for precipitation of the GST protein with glutathione-

sepharose (GS). A similar study was conducted between 100 nM GST-RGSZ2 and

HINT1. ІІ) The RGSZ2, PKCγ and HINT1 form a ternary complex: 200 nM

HINT1 were incubated with 100 nM GST-RGSZ2 in the presence of 100 nM

ZnCl2 and increasing concentrations of PKCγ. Details as in (І). ІІІ) PKCγ disrupts

HINT1-RGSZ2 association in presence of GαGTPγS subunits. GST-RGSZ2 (100

nM) and HINT1 (200 nM) were incubated in the absence or presence of 100 nM

Gαi2GTPγS and of PKCγ. ІV) Effect of PKCγ on RGSZ2 and HINT1. RGSZ2 (100

nM) or HINT1 (100 nM) were incubated for 20 min at RT with 30 nM PKCγ. The

Antioxidants & Redox Signaling PKCγ-induced phosphorylation of RGSZ2 and HINT1 was evaluated using

specific anti-phospho antibodies. V) Effect of HINT1 phosphorylation on its

association with RGSZ2 and the C-terminal cytosolic region of NR1 subunit (C0-

C1-C2). Native and PKC-phosphorylated HINT1 proteins (200 nM) were

incubated with either 100 nM GST-RGSZ2 or GST-NR1. VІ) Phosphorylation of

NR1 C0-C1-C2 by PKC, and (VІІ) its association with HINT1 and σ1R. Further

details in Methods.

B, Left: Effect of progesterone (PG) and pregnenolone (PN) sulfate on the

association σ1R-NR1 C0-C1-C2. Agarose-NR1 was pre-incubated in the presence The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) of 2.5 mM CaCl2 (30 min, RT) with the σ1R (100 nM) before the addition of 30 µM This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 76

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PG or PN (30 min, RT). P indicates that agarose was recovered and washed before

the analysis of the NR1-bound σ1R through SDS-PAGE and western blotting

(WB). Each bar represents the mean ± SEM of three determinations using

different gels and blots. *Significant differences with respect the control without

neurosteroid, ANOVA-Student-Newman-Keuls test; p<0.05. Right: Influence of

increasing concentrations of PG on the association of NR1 with σ1Rs. Agarose-

NR1 was incubated with the σ1R before the addition of PG. Agarose-NR1 carrying

the associated σ1Rs was recovered and washed before the addition of 200 nM

HINT1 or 100 nM CaM (in the presence of 2.5 mM CaCl2 and of 30 µM peptide

4). Low: This assay was also conducted with σ1R antagonists (S1RA and BD1047)

and the agonist PRE084 in the absence or presence of CaM.

Diagram: S1RA removes the σ1R from the NR1 subunit, favoring the binding of

Ca2+-CaM, which impairs the interaction of NMDARs with MOR-HINT1

complexes. As a result, morphine only recruits a fraction of the NMDAR activity

that is required to control its effects. (To see this illustration in color the reader is

Antioxidants & Redox Signaling referred to the web version of this article at www.liebertonline.com/ars). The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 77

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78 Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 78

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Figure 8.- The MOR-NMDAR cross-regulation requires NO and zinc metabolism:

the role of σ1Rs.

A, Because σ1R-/- mice display enhanced antinociception to opioids, the effect of

the direct activation of NMDARs by icv-injection of NMDA and of NOS inhibition,

3 nmol morphine was studied instead of 10 nmol used in wild-type mice. Saline or

50 pmol NMDA, 7 nmol L-NNA were icv-injected at 20 min before morphine

treatment into wild-type and σ1R-/- mice, and analgesia was determined using in

the warm water tail-flick test. *Significantly different from the group that received

saline and morphine, ANOVA-Student-Newman-Keuls test, p<0.05.

B, NOS provides the zinc ions that are required for the recruitment of PKCγ to the

HINT1 protein in the MOR environment. PAG synaptosomes obtained from wild-

type mice with and without in vivo inhibition of NOS, σ1R-/- mice, and HINT1-/-

mice were incubated for 4 h at 4°C with ZnCl2, the NO generator SNAP, and the

metal ion chelator TPEN. Subsequently, free zinc ions were removed by

centrifugation and extensive washing. The synaptosomal membranes were then

Antioxidants & Redox Signaling solubilized and incubated with affinity-purified IgGs raised against extracellular

sequences in MOR. The MOR-associated proteins were then separated by SDS-

PAGE and analyzed by western blotting. Doses of reactives and the incubation

time were taken from (51; 54). IP signifies immunoprecipitation and WB western

blot analysis.

C, Rescue of morphine acute analgesic tolerance via inhibition of NMDAR, NOS

or PKC. A morphine priming dose of 10 nmol was icv-injected, and analgesia

evaluated 30 min later in the “tail-flick” test. The non-competitive NMDAR

antagonist MK801 (1 nmol), the NOS inhibitors L-NNA (7 nmol) and L-NAME (20 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) nmol), or the PKC inhibitor Gö7874 (1 nmol), were icv-injected 30 min before This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 79

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administering 24 h later an identical morphine test dose of 10 nmol, and analgesia

was again measured 30 min later. Each bar indicates the mean ± SEM of the

before the morphine test dose, p<0.05.

Recovery from morphine acute antinociceptive tolerance produced by a single dose

of 10 nmol morphine in wild-type and σ1R-/- mice: effect of the antagonist of σ1Rs,

S1RA. After icv-injecting all of the mice with the morphine priming dose of 10

nmol, the analgesia was evaluated in the thermal tail-flick 30 min later. At the time

intervals that are indicated in the figure a different group of 4 mice received a test

dose of 10 nmol morphine, and analgesia was subsequently evaluated 30 min later.

*Significantly different from the control group that received the priming dose of

morphine, ANOVA, Student-Newman-Keuls test, p<0.05. In a parallel assay, the

mice received 1 nmol S1RA 30 min before the morphine priming dose. The

evaluation of antinociceptive tolerance was as described above. *Significantly

different from the control group that received S1RA and the priming dose of

Antioxidants & Redox Signaling morphine, p<0.05. The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 80

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Figure 9.- The σ1R connects MOR with the negative regulation of NMDAR

A, Presence of NR1 subunits, HINT1 and σ1R in the synaptosomes of PAG

obtained from wild-type and σ1R-/- mice. IP: MOR or NR1 was

immunoprecipitated, and the co-precipitated proteins were detected by western

Student-Newman-Keuls test, p<0.05. WT (wild-type mice), KO (σ1R-/- mice) and

P2 (synaptosomal fraction).

B, Morphine promotes little activation of the NMDAR-CaMKII pathway in σ1R-/-

mice. Mice were icv-injected with 10 nmol morphine, and ex vivo determinations

were performed at the indicated post-opioid time intervals. In σ1R-/- mice, MOR

binding to NR1 subunits was impaired, and then morphine hardly altered the

association that remained. Moreover, in the absence of σ1Rs, morphine recruited

little CaMKII activity and only for a short interval. Details in Fig. 5C and

Supplemental Fig. 4.

C, The icv-injection of the recombinant σ1R in σ1R-/- mice reduced the

Antioxidants & Redox Signaling antinociceptive potency of morphine. The mice were icv-injected with 0.5 nmol

recombinant σ1R, and the effect of morphine was evaluated 24 h later.

*Significantly different from the group that received saline instead of the σ1R,

p<0.05. The icv-injection of the σ1R restored the NMDAR negative regulation on

MORs. The σ1R-/- mice that had received the σ1R were icv-injected with 50 pmol

NMDA and 10 nmol

that received saline instead of NMDA, p<0.05. Inset: Solubilized brain membranes

from σ1R-/- mice were incubated with biotinylated IgGs directed against the NR1.

After recovery with streptavidin-sepharose (P), the NR1-containing complexes The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) were exposed to σ1R recombinant protein (200 nM) before detecting HINT1- This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 82

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associated proteins by SDS-PAGE chromatography and western-blotting (WB).

This study was repeated at least twice using different preparations. Equal loading

was determined from the NR1 signal. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 83

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Figure 10.- The σ1R antagonist S1RA transfers HINT1 proteins from morphine-

activated MORs to NMDAR NR1 subunits.

A, Groups of 48 mice each received an icv injection of only morphine or 3 nmol

S1RA 10 min, 30 min, 1 h, 3 h or 24 h before treatment with 10 nmol morphine. A

total of 8 mice from each group were sacrificed 90 min, 6 h or 24 h after opioid

treatment. Control mice received saline instead of S1RA or morphine. The MORs

were immunoprecipitated (IP) from PAG synaptosomes, and the co-precipitated

NR1 subunits were immunodetected by western blotting (WB). *Significantly

different from the respective control group that received saline instead of

morphine, p<0.05.

Association of HINT1 with MORs and NR1 subunits. In the experimental groups

described above, the association of HINT1 proteins with MORs and NR1 subunits

was determined for control, morphine- and S1RA-morphine-treated groups 90

min and 24 h post-morphine treatment. The circle shows that morphine alone

induces the transfer of HINT1 to NR1 subunits. The S1RA-morphine intervals that

Antioxidants & Redox Signaling did not enhance analgesia but conferred protection from tolerance are framed.

Controls of MOR and NR1 immunoprecipitation are shown in Supplemental Fig.

The diagrams represent the framed S1RA-morphine intervals. S1RA administered

1 h or 24 before morphine did not enhance analgesia or alter the MOR-promoted

NMDAR activity at 24 h, as indicated by NMDAR (****) (Fig. 5A & 6A).

However, some protection against analgesic tolerance was observed (Fig. 6B).

Upon the S1RA-mediated removal of σ1Rs, the NR1 subunit binds tightly to

HINT1 at the MOR C terminus. HINT1 carries the activated PKCγ that acts on The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) NR1 C1 S890/896, promoting the separation of the MOR C terminus from NR1- This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 84

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HINT1. Under these circumstances, CaMKII and PKC barely reach the MOR,

and then analgesic tolerance to morphine develops at a slower rate (6; 9; 48).

B, Native or mutated NR1 T879A (100 nM) binds similarly to HINT1 (200 nM).

While PKC-phosphorylated native NR1 displayed poor affinity for HINT1, PKC-

phosphorylated NR1 T879A bound to the HINT1 protein. The phosphorylation of

either form of NR1 greatly augmented its association with σ1Rs. (To see this

illustration in color the reader is referred to the web version of this article at

www.liebertonline.com/ars).

SUPPLEMENTAL DATA

Supplemental Materials and Methods

Expression of recombinant proteins

The KRX/pFN2A-NR1 (segments C0-C1-C2; NM_008169.3), KRX/pFN2A-HINT1

(NM_00824) and KRX/pFN2A-RGSZ2 (NM_001161822) strains were obtained as Antioxidants & Redox Signaling previously described (12; 13). Murine full-length 1R (AF004927) and the short variant

(AB721301) were cloned after PCR amplification from PAG cDNA into the pFN2A

(GST) Flexi vector (Promega, Spain). Specific primers containing upstream Sgf I and

downstream Pme I restriction sites were used. The PCR products were cloned

downstream of the GST coding sequence and the TEV protease site. The sequenced

proteins were identical to the GenBank™ sequences. The vector was introduced into E.

coli BL21 (KRX #L3002, Promega), and clones were selected on solid medium

containing ampicillin. After overnight induction at room temperature (1 mM IPTG and

0.1% Rhamnose), the cells were collected by centrifugation, and the pellets were The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) maintained at -80 °C. The purification of GST fusion proteins was improved using This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 85

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SUMO1-agarose (#UL-740, Boston Biochem, USA), which binds the SIM domain of

1R, this was followed by purification under native conditions on GStrap FF columns

(GE Healthcare, 17-5130-01) and when necessary the fusion proteins retained were

cleaved on the column with ProTEV protease (Promega, #V605A) and further

purification was achieved by SDS-PAGE followed by electroelution of the

corresponding gel band (GE 200, Hoefer Scientific Instruments, SF, CA) and

immobilized through agarose-coupled affinity-purified IgGs directed to the target

protein. Site directed mutagenesis was performed with Accuprime Pfx DNA

Polymerase (Invitrogen, Spain) using NR1 segments C0-C1-C2 as the template.

Threonine 879 was mutated to Alanine (T879A), and the amplified fragment was cloned

into pFN2A (GST) Flexi® Vector (Promega, Spain). The sequences were confirmed

through automated capillary sequencing. The protein Calmodulin (#208694) was

obtained from Calbiochem (Merck-Millipore, Spain), PKCγ was from Millipore (14-

483) and Abnova (P4756), Gαi2 subunits were from Calbiochem (#371796).

Antioxidants & Redox Signaling Drugs and intracerebroventricular injection

NMDA (#0114), MK801 (#0924), BD1063 (#0883), BD1047 (#0956), PRE084

(#0589), NE100 (#3133), clonidine (#0690) and WIN55,212-2 (#1038) were all

obtained from Tocris Bioscience (U.K.). We also used BD1063 (EST0013430.A; 1-[2-

(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydro- chloride), and the newly

synthesized σ1R antagonist S1RA (EST-52862.A; 4-[2-[[5-methyl-1-(2-naphthalenyl)-

1H-pyrazol-3-yl]oxy]ethyl] morpholine (3) obtained from Laboratorios Esteve.

Progesterone (P7556), Pregnenolone (P9129), Pregnenolone acetate (P49902),

Pregnenolone sulfate (P162) were obtained from Sigma (Spain), and Gö7874 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) (Calbiochem #365252) was obtained from Merck (Millipore). Morphine sulfate was This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 86

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purchased from Merck (Darmstadt, Germany). L-NG nitroarginine and NG-nitro-L-

arginine methyl ester were from Tocris (L-NNA, 0664; L-NAME, 0665). The

substances were each injected into the lateral ventricle of mice at 4 μL as previously

described (8). Compounds were dissolved in saline except σ1R ligands that were

prepared in ethanol:cremophor EL: saline (1:1:18). Doses of the compounds were taken

from previous studies (5; 13; 14)

Page 88 of 110

Immunohistochemistry

The mice were deeply anesthetized with chloral hydrate 28% weight/volume (0.1

mL/100 g body weight), prior to intracardially perfusing them with a physiological

solution and then with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at

4°C. The mice’s brains were removed, cut into small blocks and post-fixed for 4 h at

room temperature in the same fixative. The blocks were cryoprotected at 4° C in a 30%

sucrose solution in PB saline (PBS), snap frozen on dry ice and stored at -80°C. Coronal

cryosections (10 μm, 2800 Frigocut, Reichert-Jung) were mounted on gelatin-coated

slides and permeabilized in PBS containing 0.3% Triton X-100 (PBT) for 30 min at

room temperature. The sections were incubated with PBT containing 1% bovine serum

albumin for 1 h at room temperature and a rabbit IgG labeling kit (Zenon Tricolor

Rabbit IgG labeling Kit #1; Molecular Probes, Eugene, OR) was then used to detect the

rabbit polyclonal IgGs used, that fulfill the recommended criteria for use in

immunohistochemistry (15): anti-MOR (C-terminal region, Abcam, ab 134054; Alexa

Fluor 647) (2), anti-NMDAR NR1 subunit (C-terminus, Merk-Millipore, Chemicon

Antioxidants & Redox Signaling AB9864; Alexa Fluor 555) (11), and anti-σ1R (internal region139-157, Invitrogen 42-

3300; Alexa Fluor 488) (1). The labeling of the used antibodies was performed

according to the Zenon Complex Formation protocol and diluted in PBT to the desired

working concentration. The sections were incubated with the labeled IgGs in a

humidified chamber overnight at 4ºC, and then the sections were washed with PBS,

mounted, and examined with confocal microscopy (Leica TCS SP-5/LAS AF Lite

Software, Microsystems, GmbH). DAPI (4′-6-diamidino-2-phenylindole) nuclear

counterstaining was performed to localize the cells. For each channel, the first 4

microphotographs at a distance of 1 µm apart were Z-merged and the background was The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) subtracted to enhance the specificity and visibility of the signals (ImageJ, NIH, This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 88

Page 89 of 110

Bethesda, Maryland) (16). Controls for immunohistochemistry were performed

following standard protocols (for further details see Supplemental Fig. 3).

Immunoprecipitation and western blotting

After icv morphine, groups of eight mice were killed at various intervals post-opioid;

PAG were obtained and processed to obtain the synaptosomal pellet as previously

described (13), and used for MOR and NR1 immunoprecipitation and co-precipitation

of HINT1, NR1 and MOR. This procedure has been described elsewhere (4; 7). Briefly,

the PAGs were collected and homogenized in 10 volumes of 25 mM Tris-HCl pH 7.4,

and 0.32 M sucrose supplemented with a phosphatase inhibitor mixture (P2850, Sigma),

H89 (B1427, Sigma) and a protease inhibitor cocktail (P8340, Sigma). The homogenate

was centrifuged at 1000xg for 10 min to remove the nuclear fraction. The supernatant

(S1) was centrifuged twice at 20,000xg for 20 min to obtain the crude synaptosomal

pellet (P2). The final pellet was diluted in Tris buffer supplemented with a mixture of

protease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 2 μg/mL leupeptin, and

Antioxidants & Redox Signaling 0.5 μg/mL aprotinin), followed by division into aliquots and freezing at -80 ºC.

For immunoprecipitation studies, the PAG from 8 mice were typically pooled.

The assays were repeated at least twice on samples receiving an identical treatment and

collected at the same interval post-administration. The affinity purified IgGs against the

extracellular domains of the MOR 2EL (205-216: MATTKYRQGSID; GenScript Co.,

USA) and the NMDAR NR1 subunit (483-496: KFGTQERVNNSNKK; GenScript Co)

were labeled with biotin (Pierce #21217 & 21339). Pilot assays were performed to

optimize the amount of IgG and sample protein needed to precipitate the desired protein

in a single run. Target proteins were subsequently immunoprecipitated from solubilized The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) membranes and resolved through SDS/polyacrylamide gel electrophoresis (PAGE) as This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 89

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previously described (13). The Nonidet P-40 solubilized proteins were incubated

overnight at 4ºC with biotin-conjugated primary antibodies raised against the target

protein, e.g., murine MOR or NR1 subunits. The immunocomplexes were recovered

and resolved by SDS-polyacrylamide gel electrophoresis (PAGE). HINT1 was resolved

in 4–12% Bis–Tris gels (Invitrogen, NuPAGE NP0341) with NuPAGE MES SDS

running buffer (Invitrogen, NP0002) and SeeBlue Plus2 prestained Standards

(Invitrogen LC5925; 3–188 kDa). Sufficient protein was obtained by

immunoprecipitation to load about four gel lanes.

The separated proteins were then transferred onto 0.2 μm polyvinylidene difluoride

(PVDF) membranes (BioRad 162-0176. Spain) and probed overnight at 6°C with the

selected primary antibodies diluted in Tris-buffered saline pH 7.7 (TBS) + 0.05%

Tween 20 (TTBS) in DecaProbe chambers (PR 150, Hoefer-GE, Spain). Those were

detected using secondary antibodies conjugated to horseradish peroxidase. Antibody

binding was visualized by chemiluminescence (#170-5061, BioRad) and recorded using

a ChemiImager IS-5500 (Alpha Innotech, San Leandro, California). Densitometry was

Antioxidants & Redox Signaling performed using Quantity One Software (BioRad), and expressed as the mean ± SEM of

the integrated volume (average optical density of the pixels within the object area/mm2).

Immunosignals are shown relative to 0 min (animals not receiving the opioid were

attributed an arbitrary value of 1). Equal loading among the post-opioid intervals was

verified after determining the target protein in parallel blots of the same

immunoprecipitated samples used to study co-precipitation. Each bar is the mean ±

SEM of three assays performed on PAG samples obtained from independent groups

mice.

Page 91 of 110

Effect of zinc ions and NO generators on the recruitment of PKCγ to PAG MORs

in synaptosomal membranes

The effect of zinc ions, the NO generator (5)-Nitroso-N-acetylpenicillamine (SNAP;

(Tocris bioscience #0598), and the metal ion chelator N’,N’,N’,N’-tetrakis(2-

pyridylmethyl) ethylenediamine (TPEN; Fluka WA16827)) on the association of PKCγ

with MORs was studied in PAG synaptosomal membranes. Membranes were incubated

with zinc chloride (Puratronic, Alfa Aesar 231-592-0) for 4 h at 4°C before the MORs

were immunoprecipitated. Subsequently, free zinc ions were removed by centrifugation

and extensive washing. The synaptosomal membranes were then solubilized in Nonidet

P-40 buffer as described (13). The solubilized membranes were incubated overnight at

4°C with approximately 3 μg of a biotin (Pierce #21217 and 21339) conjugated primary

antibody (affinity-purified IgGs) raised against extracellular sequences in MOR. The

MOR-associated proteins were then separated by SDS-PAGE and analyzed by Western

blotting.

Antioxidants & Redox Signaling Antibodies

The primary antibodies included a newly synthesized anti-σ1R against the murine

peptide sequence SRLIVELRRLHPGH (amino acids 59–72, GenScript) exhibiting

<10% homology to other known protein sequences (EMBL, GenBank and SwissProt

databases). Anti-σ1R IgGs were purified by affinity chromatography using the antigenic

peptide coupled to NHS-activated Sepharose 4 Fast Flow (#17-0906-01, GE). Other

antibodies included anti-σ1R (AB2 #42-3300, Invitrogen); anti-MOR CT (13); anti-

NMDAR1 (#MAB1586, Merk-Millipore); anti-NMDAR1 phospho-Ser890 (#3381, Cell

Signaling), anti-NMDAR1 phospho-Ser896 (#ABN88, Merk-Millipore); anti-NMDAR1 The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) phospho-Ser897 (#ABN99, Merk-Millipore); anti-NMDAR2A phospho Y1325 This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 91

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(#ab16646, Abcam); anti-NMDAR2B phospho Y1472 (#ab59205, Abcam); anti-

CAMKIIpan (#3362, Cell Signaling); anti-Phospho-Ca2+/calmodulin-dependent protein

kinase IIα (#3361, Cell Signaling); anti-calmodulin (#4830, Cell Signaling); anti-

PKCI/HINT1 (#H00003094-A01, Abnova). Anti-RGSZ2 was raised against aa 192-

215, GenScript (6); Anti-PKC (#ab4145, Abcam); anti-Actin (#CSA-400, Stressgen);

Phosphoserine detection kit (clones 1C8, 4A9 and 16B4; #525282, Calbiochem);

Phosphothreonine detection kit (#525288, Calbiochem).

Page 93 of 110

Supplemental Figure 1

The σ1R Sumo Interacting Motif. The σ1R loop region contains a SIM (LIVEL, 61-65)

(9; 10). Both forms of the σ1R could be purified from the bacterial extract with agarose-

SUMO 1, 2 or 3. The image corresponds to the GST-σ1R and total protein was revealed

by fluorescence (SYPRO Ruby, Invitrogen) and captured in an Alpha-Innotech

(ChemiImager 5500; Santa Clara, CA 95051). Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 93

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Antioxidants & Redox Signaling

Supplemental Figure 2

Interference assay of the association between the σ1R loop region and the cytosolic The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993)

region of NMDAR NR1 subunits. A series of overlapping peptides (30 µM) mapping the This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 94

Page 95 of 110

loop region of σ1R were incubated with 100 nM NR1 C0-C1-C2 subunits prior to the

addition of 100 nM σ1R. GST-NR1 was precipitated, and the associated protein was

evaluated through ECL-densitometry (Alpha-Innotech, ChemiImager 5500-AlphaEase).

These data were obtained from three independent assays. *Significantly different from

the σ1R signals observed in the absence of interfering peptides, ANOVA-Student-Keuls

test; p<0.05. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 95

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96 Antioxidants & Redox Signaling

Supplemental Figure 3

Co-localization of the NMDAR NR1 subunit with σ1R and the MOR, and of σ1R with

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) the MOR in mouse PAG neurons. This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 96

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A. Left panel, Original Cajal drawing showing a view of the ventro-lateral region of

PAG, Golgi method. A: cerebral aqueduct; B & C axons in Darkschewitsch nucleus; E,

fiber-rich region of the central gray matter. Right panel, Coronal drawing of mouse

brain showing the PAG region analyzed in this triple co-localization of σ1R, NMDAR

NR1 subunit and MOR.

B. Confocal laser-scanning microphotographs taken from coronal sections of the mouse

midbrain PAG. The individual labeling and triple co-localization of NMDAR NR1

subunit (red), σ1R (green) and MOR (blue) was studied in the dorsolateral region (red

square in the PAG diagram). Nuclei are in grey (DAPI) and the MOR, NR1, and σ1R

were observed with Alexa Fluor 647, 555 and 488, respectively. In this ventro-lateral

PAG region, triple co-localization of the MOR, σ1R and NR1 subunits is mostly

observed in fibers, probably corresponding to synapses. Note that high-power

magnification panel (nuclei were removed) shows triple co-localization as white regions

(red arrows). Coincidence between NR1 and σ1R is shown in yellow color. The MOR

co-localizes with σ1R (purple color) and NR1 (light green color). For details see

Antioxidants & Redox Signaling Methods and Fig. 4.

A positive control sample labeled with conventional secondary detection methods was

included in the pilot studies in order to confirm the specificity of the staining observed

with Zenon labeling. A negative control was processed with the same protocol but with

the omission of the primary antibody to assess non-specific labeling.

C. immunodetection of the MOR or of σ1R in the PAG of wild-type and knock-out

mice (both antibodies in red and the nuclei in blue). Note the negative staining for either

MOR or σ1R in the respective KO mice.

Page 98 of 110

Antioxidants & Redox Signaling

Supplemental Figure 4

Representative blots describing the effect of the σ1R antagonist S1RA on MOR-

mediated activation of NMDARs. Groups of 42 mice each received 10 nmol of

morphine alone or 3 nmol of S1RA at 30 min before opioid treatment. Subsequently, for

each group receiving morphine, 6 mice were sacrificed at the indicated intervals.

Control mice received saline instead of morphine. PAG synaptosomes were obtained to

determine the presence of CaMKII P-Thr286, NR1 C1 P-Ser890, NR2A P-Tyr 1325.

The MOR proteins were immunoprecipitated, and the associated NR1 subunits were

The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) determined through western blotting. The icv administration of 10 nmol morphine

increased the function of NMDARs. The opioid administration increased the This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 98

Page 99 of 110

phosphorylation of the NR1 (PKC on S890) and NR2 subunits (Src on Y1325).

Moreover, morphine increased the T286 autophosphorylation of the serine and

threonine kinase CaMKII. A reduction of the MOR-NR1 association, an index of MOR

recruitment of NMDAR activity, was also observed. The administration of 3 nmol of

S1RA at 30 min before morphine significantly reduced parameters of NMDAR activity.

Immunosignals (average optical density of the pixels within the object area/mm2;

Quantity One Software, BioRad) were expressed as the change relative to the Control

group (attributed an arbitrary value of 1). Each bar represents the mean ± SEM of the

data from three determinations performed in different gels-blots. For every post-opioid

interval, indicates a significant difference respect the value of the group treated with

only morphine, ANOVA-Student-Newman-Keuls test; p<0.05. We observed no

differences in the levels of the non phosphorylated proteins. To increase readability, the

frames indicate higher exposition of the blots. Because the groups in the comparison are

equally affected through this enhancement, the result remains unaltered. IP signifies

immunoprecipitation and WB western blot analysis. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 99

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100 Antioxidants & Redox Signaling

Supplemental Figure 5

Effect of in vivo NOS inhibition on MOR-mediated activation of NMDARs. The mice

received saline or 7 nmol L-NNA (an inhibitor of NOS) 30 min before the icv-injection

of 10 nmol morphine, subsequently 6 mice were sacrificed at the indicated intervals.

PAG synaptosomes were obtained and solubilized as described in Methods. The MOR The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) proteins were immunoprecipitated, and the associated NR1 subunits, PKCγ and HINT1 This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 100

Page 101 of 110

101

proteins were determined through western blotting. The presence of NR1 C1 P-Ser890,

NR2A P-Tyr 1325, CaMKII P-Thr286 and total CaMKII was determined through SDS-

PAGE followed by western blotting analysis of PAG synaptosomes. Immunosignals

(average optical density of the pixels within the object area/mm2; Quantity One

Software, BioRad) were expressed as the change relative to the Control group

(attributed an arbitrary value of 1, dashed line). Each bar represents the mean ± SEM of

the data from three determinations performed in different gels-blots. Indicates a

significant difference respect the value of the corresponding control group, ANOVA-

Student-Newman-Keuls test; p<0.05. We observed no differences in the levels of the

non phosphorylated proteins. IP signifies immunoprecipitation and WB western blot

analysis.

Page 102 of 110

102

Supplemental Figure 6 Antioxidants & Redox Signaling The absence of σ1Rs disconnects NMDAR negative influence from MOR signaling.

-/- A, Morphine produces heterologous tolerance in σ1R mice. Groups of four mice each

were injected icv with 10 nmol morphine and the analgesic effect of morphine, 150

nmol clonidine, and 20 nmol WIN55,212-2 was assessed 24 h later (30 min after

morphine and the agonist of α2-adrenoceptors clonidine, 10 min after the cannabinoid) .

*Significantly different from the respective control group that received the priming dose

of morphine, ANOVA, Student-Newman-Keuls test, p<0.05.

-/- B, Morphine promotes little activation of the NMDAR-CaMKII pathway in σ1R The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) mice: effect of S1RA. Mice were icv-injected with 10 nmol morphine and the ex vivo This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 102

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103

assays were performed at the post-opioid time intervals indicated. The administration of

-/- S1RA to σ1R mice did not alter these molecular effects of morphine. IP signifies

immunoprecipitation and WB western blot analysis.

Page 104 of 110

104

Supplemental Figure 7

Controls of MOR and NR1 immunoprecipitation -see Fig. 9. MORs and NR1 subunits

were determined through western blot analysis for control, morphine and various

S1RA-morphine treated groups and sacrificed at 90 min, 6 h and 24 h post-morphine. IP

signifies immunoprecipitation.

Page 105 of 110

105 Antioxidants & Redox Signaling

Supplemental Figure 8

Antagonists of σ1Rs promote the uncoupling of MOR activity from the negative control

of glutamate NMDARs. Antagonists of σ1Rs promote the uncoupling of MOR activity

from the negative control of glutamate NMDARs. Antagonists of σ1Rs such as S1RA

facilitate NR1 C0 binding with Ca2+-CaM to inhibit NMDAR calcium fluxes, indicated The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) by the framed NMDAR without asterisks. In the absence of MOR regulation of PLCs, This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 105

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106

Ca2+-CaM binding further reduces calcium levels in the NMDAR environment

promoting the subsequent release of calcium-depleted CaM from NR1 subunits. In these

circumstances, MOR-HINT1 binds with high affinity to NR1 subunits being observed

as a time-dependent increase in MOR-NR1 association. In PAG synaptosomes derived

from mice treated with only S1RA, the σ1R antagonist produced no other noticeable

changes in the other molecular parameters evaluated. IP signifies immunoprecipitation

and WB western blot analysis. Antioxidants & Redox Signaling The 1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA negative control (doi: 10.1089/ars.2014.5993) This article has been peer-reviewed and accepted for publication, but yet to undergo copyediting proof correction. The final published version may differ from this proof. 106

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107

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