Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

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SignificanceAgonists of the μ-opioid receptor (MOPr) are currently the gold standard for pain treatment. However, their therapeutic usage is greatly limited by side effects including respiratory depression, constipation, tolerance, and dependence. Functionally selective MOPr agonists that mediate their effects preferentially through G proteins rather than β-arrestin signaling are believed to produce fewer side effects. Here, we present the discovery of 3 unusual tetrapeptides with a unique stereochemical arrangement of hydrophobic amino acids from an Australian estuarine isolate of Penicillium species. Building on these natural templates we developed bilorphin, a potent and selective highly G protein-biased agonist of the MOPr. Further, through the addition of a simple sugar moiety, we generated bilactorphin that is an effective analgesic in vivo.

Ionic currents can be evoked by mechanical inputs applied directly at the cell-substrate interface. These ionic currents are mediated by mechanically activated ion channels, where the open probability increases with increasing mechanical input. In order to study mechanically activated ion channels directly at the interface between cells and their environment, we have developed a technique to simultaneously monitor ion channel activity whilst stimuli are applied via displacement of cell-substrate contacts. This technique utilizes whole-cell patch-clamp electrophysiology and elastomeric pillar arrays, it is quantitative and appropriate for studying channels that respond to stimuli that are propagated to an adherent cell via the physical substrate. The mammalian channels PIEZO1, PIEZO2 have been shown to be activated by substrate deflections, using this technique. In addition, TRPV4 mediated currents can be evoked by substrate deflections, in contrast to alternate stimulation methods such as membrane stretch or cellular indentation. The deflections applied at cell-substrate points mimic the magnitude of physical stimuli that impact cells in situ .

Ion channels activated by mechanical inputs are important force sensing molecules in a wide array of mammalian cells and tissues. The transient receptor potential channel, TRPV4, is a polymodal, nonselective cation channel that can be activated by mechanical inputs but only if stimuli are applied directly at the interface between cells and their substrate, making this molecule a context-dependent force sensor. However, it remains unclear how TRPV4 is activated by mechanical inputs at the cell-substrate interface, which cell intrinsic and cell extrinsic parameters might modulate the mechanical activation of the channel and how mechanical activation differs from TRPV4 gating in response to other stimuli. Here we investigated the impact of substrate mechanics and cytoskeletal components on mechanically evoked TRPV4 currents and addressed how point mutations associated with TRPV4 phosphorylation and arthropathy influence mechanical activation of the channel. Our findings reveal distinct regulatory modulation of TRPV4 from the mechanically activated ion channel PIEZO1, suggesting the mechanosensitivity of these two channels is tuned in response to different parameters. Moreover, our data demonstrate that the effect of point mutations in TRPV4 on channel activation are profoundly dependent on the gating stimulus.

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