The existence and importance of the neuronal opioid signaling system in central nervous system (CNS) biology are well established and documented in basic research and clinical practice. A family of receptors and endogenous ligands form the molecular basis of this system and an extensive literature has documented their roles in the normal control of CNS cellular and system biology. Moreover, it has been well established that CNS opioid receptors are primary targets of therapeutic agents and abused drugs, which has lead to significant interest in an understanding of receptor structure, function and pharmacological profile. The mu opioid receptor (MOR) is of particular interest because of its central role in therapeutic control of pain and in narcotic addiction. A number of polymorphisms in the human gene (OPRM1) for MOR have been identified and emerging research suggests that MOR variants may play a role in the well-documented inter-individual variability in the sensitivity of humans to drugs that act at MOR. Of particular interest is the OPRM1 A118G variant, which generates a single amino-acid substitution in the N-terminal domain of MOR (Asn40Asp) and codes for 40Asp mutant receptors (N40D-variant). Central to an understanding of the functional consequences of this mutation is an identification of functional changes in MOR signaling due to the mutation. A critical aspect of receptor signaling is receptor dynamics, which determines receptor availability, function and the downstream consequences of receptor activation. Recent advances in molecular imaging now offer a new approach to study receptor dynamics, Fluorescence Correlation Spectroscopy (FCS) integrated with Confocal Laser Scanning Microscopy (CLSM). FCS/CLSM enables nondestructive observation of molecular interactions in living cells in real time with ultimate single-molecule sensitivity. We propose to utilize this new methodology and parallel physiological analyses to study the cellular dynamics of wild type and N40D-variant and the functional consequence of these interactions to neuronal physiology. We hypothesize that the N40D-variant will show altered receptor dynamics that are ligand dependent and that the altered receptor dynamics will result in downstream alterations in receptor coupling to neurophysiology.
Three Specific Aims are proposed: (1) To investigate at the molecular and cellular level the potential differences in functional dynamics between the wild type human MOR and its N40D variant. (2) To identify differences in the downstream functional consequences of MOR and N40D-variant opioid receptor dynamics in live cells expressing these receptors using electrophysiological and Ca2+ imaging techniques. (3) To extend studies of MOR and N40D-variant to native neurons (cultured hippocampal neurons) where investigations of receptor dynamics and functional coupling can be pursued in live cells within neuronal circuits.
Accumulating evidence suggests that the normal occurrence of mutations in the human mu opioid receptor has consequences for opioid analgesia and drug addiction in humans expressing the variant form. Our understanding of how these mutations affect mu opioid receptor function is limited. To fill this gap in our knowledge we will use a newly developed approach to study the functional properties in both the normal mu opioid receptor and a variant that is commonly expressed in the population and has been implicated in altered sensitivity to drugs that act at the mu receptor.
| Gruol, Donna L; Nelson, Thomas E; Hao, Christine et al. (2012) Ethanol alters opioid regulation of Ca(2+) influx through L-type Ca(2+) channels in PC12 cells. Alcohol Clin Exp Res 36:443-56 |
