Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors.

Despina Giakomidi,David G Lambert,John Mcdonald,Mark F Bird,Erika Marzola,Nidhuna Sabu,Serena Chanoch,Remo Guerrini,Barbara Horley,Girolamo Calo

doi:10.1371/journal.pone.0250011

Abstract

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

Highlights

Opioid receptors are members of the seven transmembrane-spanning G protein-coupled receptor (GPCR) superfamily
40μg of freshly prepared membrane protein was suspended in 0.5ml of buffer containing 50mM Tris, 0.5% BSA and ~0.8nM [3H]-Diprenorphine (DPN) or ~0.8nM [3H]-Nociceptin/Orphanin FQ (N/OFQ) and varying concentrations (1pM-1μM) of unlabelled Dermorphin or DermATTO488
Coverslip cultures of Chinese Hamster Ovary (CHO) or Human Embryonic Kidney (HEK) cells were placed on a Harvard Peltier plate and perfused with Krebs buffer, pH 7.4 at 4 ̊C

Summary

Introduction

Opioid receptors are members of the seven transmembrane-spanning G protein-coupled receptor (GPCR) superfamily. MOP receptors couple to Gi/Go G-proteins to enhance an outward potassium conductance to hyperpolarize, close voltage-sensitive calcium channels and inhibit adenylyl cyclase leading to the reduction of cAMP formation. In neurones this leads to reduced firing and neurotransmitter release [1,2,3,4,5]. Dermorphin binds to MOP with high affinity and an order of magnitude selectivity over DOP [14] This relatively short (seven amino acids) peptide is easy to manipulate so we have used it as an acceptor for the fluorescent ATTO dye (488nm) to produce [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We use DermATTO488 to visualise MOP expression in live CHO and HEK cells using confocal microscopy

Materials and methods

Results

Discussion