SUMMARY Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency, illustrating a new paradigm in toxin structure and evolution. The ‘double-knot’ toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.

The AMPA-type glutamate receptors mediate the majority of the fast excitatory synaptic transmission and critically contribute to synaptic plasticity in the brain, hence the existence of numerous trafficking proteins dedicated to regulation of their synaptic delivery and turnover. Stargazin (also termed ␥2) is a member of a recently identified protein family termed transmembrane AMPA receptor regulatory proteins (TARPs). TARPs physically associate with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. Here, we report that next to its trafficking roles, stargazin may also act as a positive allosteric modulator of AMPA receptor ion channel function. Coexpression of stargazin with AMPA receptor subunits, either in Xenopus oocytes or in human embryonic kidney 293 cells, significantly reduced receptor desensitization in response to glutamate. Receptor deactivation rates were also slowed, and the recovery from desensitization was accelerated. Structurally, based on the data showing a tight correlation between desensitization and the stability of the AMPA receptor intradimer interface, we propose that binding of stargazin may stabilize the receptor conformation. Functionally, our data suggest that AMPA receptors complexed with stargazin (and possibly also with other TARPs) at the postsynaptic membrane are significantly more responsive to synaptically released glutamate compared with AMPA receptors lacking stargazin/TARP interaction. The putative existence of such two states of synaptic AMPA receptors, with and without stargazin/TARP binding, may provide a novel mechanism for regulation of excitatory synaptic strength during development and/or in synaptic plasticity in the adult brain.

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X4 receptor channels (Khakh, B.S., W.R. Proctor, T.V. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289–7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (≤10 μM) applied from outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate of current deactivation. These two phenomena likely result from the binding of IVM to separate sites. A higher affinity site (EC50 0.25 μM) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the EC50 and Hill slope of the ATP concentration-response relationship. A lower affinity site (EC50 2 μM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X4 receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 μM) increased the number of openings per burst, without significantly changing the mean open or mean shut time within a burst. At higher concentrations (1.5 μM) of IVM, two additional open time components of long duration were observed that gave rise to long-lasting bursts of channel activity. Together, the results suggest that the binding of IVM to the higher affinity site increases current amplitude by reducing channel desensitization, whereas the binding of IVM to the lower affinity site slows the deactivation of the current predominantly by stabilizing the open conformation of the channel.

The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

A prominent feature of ionotropic glutamate receptors from the AMPA and kainate subtypes is their profound desensitization in response to glutamate-a process thought to protect the neuron from overexcitation. In AMPA receptors, it is well established that desensitization results from rearrangements of the interface formed between agonist-binding domains of adjacent subunits; however, it is unclear how this mechanism applies to kainate receptors. Here we show that stabilization of the binding domain dimer by the generation of intermolecular disulfide bonds apparently blocked desensitization of the kainate receptor GluR6. This result establishes a common desensitization mechanism in both AMPA and kainate receptors. Surprisingly, however, surface expression of these nondesensitizing mutants was drastically reduced and did not depend on channel activity. Therefore, in addition to its role at the synapse, we now propose an intracellular role for desensitization in controlling maturation and trafficking of glutamate receptors.

Altered glucose reabsorption the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CBR), which contributes to the development of diabetic nephropathy (DN). However, the link between CBR and GLUT2 remains to be determined. Here, we show that chronic peripheral CBR blockade or genetically inactivating CBRs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CBR also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CBR or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CBR antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2X cilia ) which is modulated by Na + and by long exposures to ATP. P2X cilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch-clamp technique in the whole-cell configuration. During the initial continuous exposure to extracellular ATP, P2X cilia currents gradually increase in magnitude (priming), yet the permeability to N -methyl-D-glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na + , which readily permeates P2X cilia receptor channels, was found to inhibit the channel extracellular to the electric field. Cation-selective ion channels activated by extracellular ATP (P2X receptor channels) are widely distributed in electrically excitable cells such as neurones, and in non-excitable cells such as immune cells and epithelial cells (North, 2002). Seven subunits of the mammalian P2X receptor family (P2X 1 to P2X 7 ) have been cloned, and all of them can form functional homomeric receptor channels in heterologous expression systems (North, 2002;Jones et al. 2004). Six functional heteromeric receptor channels (P2X 1 /P2X 2 , P2X 1 /P2X 4 , P2X 1 /P2X 5 , P2X 2 /P2X 3 , P2X 2 /P2X 6 and P2X 4 /P2X 6 ) have also been characterized (Torres et al. 1998;Le et al. 1998Le et al. , 1999King et al. 2000;Brown et al. 2002;Nicke et al. 2005). Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that additional hetero-oligomeric assemblies are possible among the different P2X receptor subunits (Torres et al. 1999). The pharmacological and biophysical properties of native P2X receptor channels often deviate from those of heterologously expressed P2X receptor channels. This suggests that native P2X receptor channels might be processed differently or that they are heteromeric channels of yet uncharacterized combinations of P2X subunits.Airway ciliated cells express a P2X receptor channel (P2X cilia ) of unknown composition. This channel is interesting both from a physiological and from a biophysical standpoint because of its modulation by both ATP and Na + . P2X cilia receptor currents gradually increase over tens of seconds during the first exposure to ATP whereas subsequent applications of ATP activate the current significantly faster (Korngreen et al. 1998). This priming phenomenon can be observed for several minutes after the initial application of ATP, and thus ATP can be considered both an agonist and a long-term modulator of the P2X cilia receptor channel. P2X cilia receptor channels are also modulated by Na + (Ma et al. 1999). The effect

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel’s outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation.