Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gβγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K⁺ conductance and membrane potential. TASK-1/3 activity is regulated by hormones and transmitters that act through G protein-coupled receptors (GPCR) signalling via G proteins of the Gα_q/11 subclass. How the receptors inhibit channel activity has remained unclear. Here, we show that TASK-1 and -3 channels are gated by diacylglycerol (DAG). Receptor-initiated inhibition of TASK required the activity of phospholipase C, but neither depletion of the PLC substrate PI(4,5)P₂ nor release of the downstream messengers IP₃ and Ca²⁺. Attenuation of cellular DAG transients by DAG kinase or lipase suppressed receptor-dependent inhibition, showing that the increase in cellular DAG—but not in downstream lipid metabolites—mediates channel inhibition. The findings identify DAG as the signal regulating TASK channels downstream of GPCRs and define a novel role for DAG that directly links cellular DAG dynamics to excitability.

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P₃ > PI(3,5)P₂ > PI(4,5)P₂ ≈ PI(3,4)P₂ >> PI(4)P_. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

Background and Purpose: Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122‐sensitive ion channels.Experimental Approach: We performed detailed biophysical analysis of the U73122‐induced currents in frequently used cell lines.Key Results: At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P₂and Ca²⁺. U73122 also potentiated Ca²⁺‐dependent TRPM4 currents in human Jurkat T‐cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family.Conclusions and Implications: Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.

Aminoglycoside antibiotics (AGs) are severely ototoxic. AGs cause degeneration of outer hair cells (OHCs), leading to profound and irreversible hearing loss. The underlying mechanisms are not fully understood. OHC survival critically depends on a specific K⁺ conductance (I_K,n) mediated by KCNQ4 (Kv7.4) channels. Dysfunction or genetic ablation of KCNQ4 results in OHC degeneration and deafness in mouse and humans. As a common hallmark of all KCNQ isoforms, channel activity requires phosphatidylinositol(4,5)bisphosphate [PI(4,5)P₂]. Because AGs are known to reduce PI(4,5)P₂ availability by sequestration, inhibition of KCNQ4 may be involved in the action of AGs on OHCs. Using whole-cell patch-clamp recordings from rat OHCs, we found that intracellularly applied AGs inhibit I_K,n. The inhibition results from PI(4,5)P₂ depletion indicated by fluorescence imaging of cellular PI(4,5)P₂ and the dependence of inhibition on PI(4,5)P₂ availability and on PI(4,5)P₂ affinity of recombinant KCNQ channels. Likewise, extracellularly applied AGs inhibited I_K,n and caused substantial depolarization of OHCs, after rapid accumulation in OHCs via a hair cell-specific apical entry pathway. The potency for PI(4,5)P₂ sequestration, strength of I_K,n inhibition, and resulting depolarization correlated with the known ototoxic potential of the different AGs. Thus, the inhibition of I_K,n via PI(4,5)P₂ depletion and the resulting depolarization may contribute to AG-induced OHC degeneration. The KCNQ channel openers retigabine and zinc pyrithione rescued KCNQ4/I_K,n activity from AG-induced inhibition. Pharmacological enhancement of KCNQ4 may thus offer a protective strategy against AG-induced ototoxicity and possibly other ototoxic insults.