Kenneth Lindegaard Madsen

Despite urgent need for better analgesics, drug development has been complicated by incomplete bench-to-bedside translation. A likely contributing factor is the poor connection between the experimenter-evoked pain-related measures often used in preclinical models, and the clinical symptoms, like broader functional impairment, reported by patients. Here, we asked whether vertical climbing, a species-specific elective behaviour in mice, could serve as a measure of pain-related impairment. We tested male and female mice in a wide range of pain-related models including models of post-operative pain, inflammatory injury, osteoarthritis, rheumatoid arthritis, cancer-induced bone pain, fibrous dysplasia, neuropathic pain, migraine-related pain and facial neuropathy. Vertical climbing was reduced in all models, across five different laboratories, demonstrating the robustness and consistency of this type of behaviour as a novel pain-related outcome measure. Reduced climbing correlated significantly with established pain-related measures, but its time course diverged from that of evoked mechanical allodynia, while aligned more closely with other non-evoked behaviours, like burrowing, home cage activity, wheel running or weight bearing. Pharmacological validation was bidirectional: gabapentin restored climbing in mice with neuropathic injury, and naltrexone reinstated the climbing deficit in mice that had recovered from intraplantar CFA, consistent with persistent pain held in remission by endogenous opioid signalling. Overall, our results indicate that voluntary vertical climbing is a stable, elective behaviour that provides a robust and translationally relevant measure of pain-related functional impairment in mice.

Helix 8 in G protein-coupled receptors (GPCRs) has recently been linked to receptor internalization (Schmidt et al., 2025, Sci. Adv. 11, eadv1499), but the molecular basis of this relationship remains unclear. Here, we examined a proposed mechanism in which helix 8 functions as a surface-active amphipathic element that promotes membrane binding, senses membrane curvature, and thereby facilitates GPCR trafficking. We analyzed six amphipathic peptides, including helix 8 segments from five GPCRs spanning a broad range of internalization rates, using molecular dynamics simulations and circular dichroism spectroscopy. Secondary structure in a membrane environment showed the strongest relationship with internalization, indicating that helix-forming propensity is a key determinant of trafficking behavior. Membrane binding showed a weaker association with internalization that became more apparent when secondary structure was considered. In contrast, curvature sensing, as modeled here, did not correlate positively with internalization, although all constructs favored positive curvature and curvature-related hydrophobic phase width matched the internalization trend. Together, the results suggest that, while membrane binding and curvature-related properties likely contribute to the process, the ability of helix 8 to form amphipathic helices is the principal factor underlying differential GPCR internalization. These findings support a broader model in which surface-active amphipathic motifs tune membrane protein trafficking by coupling dynamic structural propensities to membrane interactions.

Transmembrane proteins mediate essential cellular processes including signaling, transport, and ion flux. Besides their well-characterized structured domains, most contain intrinsically disordered regions, whose biological roles remain poorly understood. Evidence suggests that the functions of intrinsically disordered regions are context-dependent, a trait particularly relevant when anchored to cellular membranes. In this study, we probed peptide arrays with fluorescent liposomes to generate a high-resolution, proteome-wide map of membrane-interaction sites within intrinsically disordered regions of human transmembrane proteins. Screening 4,000 proteins, we identified membrane-interaction sites in ~60% of cases. Among these, ~63% represent amphipathic helices, while ~17% resemble cationic cell-penetrating peptides. We demonstrate that membrane-interaction motifs can influence subcellular localization and may contribute to both physiological and pathological processes. Our findings establish membrane association as a key functional aspect of intrinsically disordered regions and provide a valuable resource for discovering non-canonical regulatory mechanisms in transmembrane proteins. The resource is available at MemRIDRdb.

Neuropeptide-containing dense core vesicles (DCVs) generated in neuronal somata are circulated in axons to supply distal release sites, depending on kinesin-1, kinesin-3, and dynein, but how the motors are recruited remains unclear. Here we use proximity proteomics in the living Drosophila nervous system to identify the protein complex responsible for recruitment of kinesin-1 and dynein on DCVs. We find that the dynein and kinesin-1 adaptor Sunday driver (Syd/dJIP3/4) interact with the DCV-located GTPase Rab2 and also bind the Arl8 effector RUFY. Disrupting Rab2, Syd, RUFY, the Arl8 activator BORC, or dynein impedes retrograde DCV flux and induces axonal accumulation of immobile DCVs. Our data suggest that dynein is recruited and activated by a Syd/RUFY complex anchored to DCVs by Rab2 and Arl8. Rab2 loss but not disruption of Syd, RUFY, or dynein causes missorting of DCV membrane proteins into vesicle aggregates in motor neuron somata, suggesting that Rab2 employs separate effectors in DCV biogenesis and motility.

Summary: Transmembrane proteins mediate essential cellular processes including signaling, transport, and ion flux. Besides their well-characterized structured domains, most contain intrinsically disordered regions, whose biological roles remain poorly understood. Evidence suggests that the functions of intrinsically disordered regions are context-dependent, a trait particularly relevant when anchored to cellular membranes. In this study, we probed peptide arrays with fluorescent liposomes to generate a high-resolution, proteome-wide map of membrane-interaction sites within intrinsically disordered regions of human transmembrane proteins. Screening 4,000 proteins, we identified membrane-interaction sites in ∼60% of cases. Among these, ∼63% represent amphipathic helices, while ∼17% resemble cationic cell-penetrating peptides. We demonstrate that membrane-interaction motifs can influence subcellular localization and may contribute to both physiological and pathological processes. Our findings establish membrane association as a key functional aspect of intrinsically disordered regions and provide a valuable resource for discovering non-canonical regulatory mechanisms in transmembrane proteins. The resource is available at MemRIDRdb .

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity-dependent internalization, which is orchestrated by adaptor proteins recognizing semispecific motifs within the receptors’ intracellular regions. Here, we describe a complementary trafficking mechanism for G protein–coupled receptors (GPCRs) that is evolutionary conserved and refined. This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a principle of receptor-lipid interactions that may have broad implications for cellular signaling and therapeutic targeting.

Glutamate receptor-dependent cytosolic acidification can be induced in hippocampal neurons by pharmacological or seizure-like stimulation. This acidification is thought to arise from Ca ²⁺ and metabolism-related processes, however, the exact underlying mechanism as well as its functional role remains uncertain. To reassess the mechanism of cytosolic acidification in excitatory hippocampal neurons and address the physiological relevance of the phenomenon, we combined pH/Ca ²⁺ biosensors to study activity-induced pH dynamics in hippocampal neurons. First, we addressed cytosolic acidification in relation to LTP at hippocampal CA3-CA1 synapses. Using hippocampal slices from adult rats of both sexes, we show that LTP-inducing stimulation at the Schaffer collaterals evokes transient cytosolic acidification in hippocampal CA1 neurons. This highlights neuronal pH shifts as a trait of general hippocampal neurotransmission rather than a marker of excitotoxicity, possibly serving as a secondary messenger. Moreover, using dissociated hippocampal neurons from rat embryos, we show that glutamate receptor agonists typically induce larger cytosolic acid shifts compared to simple depolarization or spontaneous activity, suggesting that glutamate receptor-mediated acidification involves several separate mechanisms; pyruvate-dependent dampening of neuronal acidification may reflect a direct inhibition of NMDA receptors rather than reduced glycolytic activity, questioning the previously reported involvement of metabolism in cytosolic acidification; and whereas acid shifts induced by simple depolarization show exclusive dependence on cytosolic Ca ²⁺ , AMPA-induced acidification depends both on cytosolic Ca ²⁺ and on an inward electrochemical driving force for protons. These results suggest that glutamate receptor-induced cytosolic acidification relies both on cytosolic Ca ²⁺ and on a passive proton influx, possibly mediated by the receptor itself. Graphical Significance statement: Although several studies report that hippocampal pyramidal neurons show significant cytosolic acidification in response to activation by drugs, epilepsy and stroke, the molecular mechanism and functional impact of the phenomenon remains uncertain. Using live imaging of both Ca and H dynamics, we demonstrate that the induction of LTP at hippocampal synapses is associated with cytosolic acidification in the postsynaptic neurons, suggesting that the cytosolic acid shifts may be a general trait of neurotransmission and related plasticity. We further revisit the mechanistic relation between the two ion systems. Our results suggest that glutamate receptor-induced cytosolic acidification involves two distinct mechanisms, one related to cytosolic Ca and another involving a passive H influx, possibly mediated by the receptor itself.

Chronic pain is a complex, debilitating, and escalating health problem worldwide, impacting 1 in 5 adults. Current treatment is compromised by dose-limiting side effects, including high abuse liability, loss of ability to function socially and professionally, fatigue, drowsiness, and apathy. PICK1 has emerged as a promising target for the treatment of chronic pain conditions. Here, we developed and characterized a cell-permeable fatty acid–conjugated bivalent peptide inhibitor of PICK1 and assessed its effects on acute and chronic pain. The myristoylated PICK1 inhibitor, myr-NPEG ₄ -(HWLKV) ₂ (mPD5), self-assembled into core-shell micelles that provided favorable pharmacodynamic properties and relieved evoked mechanical and thermal hypersensitivity as well as ongoing hypersensitivity and anxiodepressive symptoms in mouse models of neuropathic and inflammatory pain following subcutaneous administration. No overt side effects were associated with mPD5 administration, and it had no effect on acute nociception. Finally, neuropathic pain was relieved far into the chronic phase (18 weeks after spared nerve injury surgery) and while the effect of a single injection ceased after a few hours, repeated administration provided pain relief lasting up to 20 hours after the last injection.

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity dependent internalization, which is orchestrated by adaptor proteins recognizing semi-specific motifs within the receptors’ intracellular regions. Here we describe a complementary and evolutionary conserved and refined trafficking mechanism for G-protein coupled receptors (GPCR). This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a new principle of receptor-lipid interactions that may have broader implications for cellular signaling and therapeutic targeting. One-Sentence Summary: Receptor proteins navigate cellular membranes by interacting with their curvature using an evolutionary conserved mechanism that relies on amphipathic helices and complements direct coupling to the endocytic protein machinery.