The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K þ 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 Ga 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)P 2 nor release of the downstream messengers IP 3 and Ca 2 þ . 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.
In contrast to pain processing neurons in the spinal cord, where the importance of chloride conductances is already well established, chloride homeostasis in primary afferent neurons has received less attention. Sensory neurons maintain high intracellular chloride concentrations through balanced activity of Na +-K +-2Cl − cotransporter 1 (NKCC1) and K +-Cl − cotransporter 2 (KCC2). Whereas in other cell types activation of chloride conductances causes hyperpolarization, activation of the same conductances in primary afferent neurons may lead to inhibitory or excitatory depolarization depending on the actual chloride reversal potential and the total amount of chloride efflux during channel or transporter activation. Dorsal root ganglion (DRG) neurons express a multitude of chloride channel types belonging to different channel families, such as ligand-gated, ionotropic γ-aminobutyric acid (GABA) or glycine receptors, Ca 2+-activated chloride channels of the anoctamin/TMEM16, bestrophin or tweetyhomolog family, CLC chloride channels and transporters, cystic fibrosis transmembrane conductance regulator (CFTR) as well as volume-regulated anion channels (VRACs). Specific chloride conductances are involved in signal transduction and amplification at the peripheral nerve terminal, contribute to excitability and action potential generation of sensory neurons, or crucially shape synaptic transmission in the spinal dorsal horn. In addition, chloride channels can be modified by a plethora of inflammatory mediators affecting them directly, via protein-protein interaction, or through signaling cascades. Since chloride channels as well as mediators that modulate chloride fluxes are regulated in pain disorders and contribute to nociceptor excitation and sensitization it is timely and important to emphasize their critical role in nociceptive primary afferents in this review.
BACKGROUND AND PURPOSESignalling 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 APPROACHWe performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. KEY RESULTSAt 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)P 2 and Ca 2+ . U73122 also potentiated Ca 2+ -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 IMPLICATIONSGiven 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. AbbreviationsCi-VSP, Ciona intestinalis voltage-sensing phosphatase; IP 3 , inositol-1,4,5-trisposphate; PI5K, PI(4)P-5-kinase; PI(4,5)P 2 , phosphatidylinositol-(4,5)-bisphosphate
The three members of the ether-à-go-go-gene-like (Elk; Kv12.1-Kv12.3) family of voltage-gated K+ channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that Kv12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to Kv12.1 and Kv12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native Kv12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for Kv12.2, appropriate genetically modified mouse models have not been described; therefore, identification of Kv12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human Kv12.1 to identify distinct properties of these channels. We found that Kv12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a “relaxed” open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related Kv11.1, mode shift was not sensitive to extracellular Na+, which allowed for discrimination between these isoforms. Sensitivity of Kv12.1 and Kv11.1 to the broad-spectrum K+ antagonist 4-aminopyridine was similar. However, 4-AP strongly activated Kv12.1 channels, but it was an inhibitor of Kv11 channels. Interestingly, the agonist of Kv11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated Kv11.1, but strongly inhibited Kv12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of Kv12.1 channels and established straightforward experimental protocols to characterize Kv12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of Kv12.1 to whole cell currents.
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