Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers.

According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl(-) channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1ß. This aptamer potentiated whole-cell Cl(-) currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

Dissecting TRPV1: lessons to be learned?

The transient receptor potential channel TRPV1 is a polymodal nociceptor. It is primarily expressed in dorsal root ganglia and peripheral sensory nerve endings, and to a much lesser extent, in the central nervous system. It has also been implicated in the functional properties of e.g. urinary and bronchial epithelia. TRPV1 has long been under intensive investigation by the pharmaceutical industry as a candidate drug target especially for pain conditions. This review summarizes the current knowledge of the molecular determinants of TRPV1 channel activation by heat, protons and capsaicin. Newly discovered heat and proton activation sites within the pore domain are discussed as well as potential consequences for drug discovery. Polymodal TRPV1 antagonists were found to cause hyperthermia in a species-dependent manner in-vivo, hence the discovery of euthermic compounds with an appropriate modality selectivity profile will be crucial for TRPV1's future as a drug target.

The biophysical and molecular basis of TRPV1 proton gating.

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.

Novel temperature activation cell-based assay on thermo-TRP ion channels.

The precise temperature control of the ABI Prism 7900HT Sequence Detection System designed for detection of fluorescence of a biological sample in real-time PCR assays (TaqMan assays) was used to activate Thermo-TRP ion channels, enabling a novel 384-/96-well plate-based assay. Functional pharmacology was verified against the temperature activation using intracellular calcium fluorescence as a measure of ion channel activity. The assay is applicable to both heterologous expression systems and dorsal root ganglia primary cells. This will benefit several analgesic drug discovery programs searching for new Thermo-TRP modulators.

Modulation of Ca2+ signaling by Na+/Ca2+ exchangers in mast cells.

Mast cells rely on Ca(2+) signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca(2+) release from internal stores and Ca(2+) entry through store-operated Ca(2+) channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na(+)/Ca(2+) exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K(+)-independent and K(+)-dependent components of Na(+)/Ca(2+) exchange. Northern blot analysis indicated the predominant expression of the K(+)-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca(2+) clearance via Na(+)/Ca(2+) exchange represented approximately 50% of the total clearance when Ca(2+) signals reached levels > or =200 nM. Ca(2+) signaling and store-operated Ca(2+) entry were strongly reduced by inverting the direction of Na(+)/Ca(2+) exchange, indicating that Na(+)/Ca(2+) exchangers normally extrude Ca(2+) ions from cytosol and prevent the Ca(2+)-dependent inactivation of store-operated Ca(2+) channels. Working in the Ca(2+) efflux mode, Na(+)/Ca(2+) exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca(2+) mobilization.

Effects of large clostridial cytotoxins on activation of RBL 2H3-hm1 mast cells indicate common and different roles of Rac in FcepsilonRI and M1-receptor signaling.

Using Rho GTPases-inhibiting clostridial cytotoxins, we showed recently in RBL cells that the GTPase Rac is involved in FcepsilonRI (high-affinity receptor for IgE) signaling and receptor-mediated calcium mobilization, including influx via calcium release-activated calcium channels. Here, we studied the role of Rho GTPases in muscarinic M1 receptor signaling in RBL 2H3-hm1 cells. Clostridium difficile toxin B, which inactivates Rho, Rac, and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac but not Rho, blocked M1-mediated exocytosis, indicating that Rac but not Rho is involved in the regulation of receptor-mediated exocytosis. Although antigen-induced FcepsilonRI stimulation caused tyrosine phosphorylation of the Rac guanine nucleotide exchange factor Vav, M1 stimulation by carbachol activated Rac independently of Vav. The Rac-inactivating toxins blocked M1 receptor-induced membrane translocation of the pleckstrin homology domain of protein kinase B, which is a phosphoinositide 3-kinase effector. The M1-induced calcium release from internal stores was not affected by toxin B; however, the subsequent calcium influx from the extracellular space was inhibited. The data suggest that besides capacitative calcium entry, the M1 signaling pathway activates further calcium entry channels with mechanisms that are not affected by the inhibition of Rac.