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Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2[X.sub.4] 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 P2[X.sub.4] (hP2[X.sub.4]) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM ([less than or equal to] 10 [micro]M) 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 (E[C.sub.50] 0.25 [micro]M) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the E[C.sub.50] and Hill slope of the ATP concentration-response relationship. A lower affinity site (E[C.sub.50] 2 [micro]M) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2[X.sub.4] receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 [micro]M) 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 [micro]M) 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. KEY WORDS: ion channel gating * allosteric regulation * purinergic receptors * ATP * patch clamp techniques

Polymorphism in TCF7L2, a component of the canonical Wnt signaling pathway, has a strong association with β-cell dysfunction and type 2 diabetes through a mechanism that has yet to be defined. β-Cells rely on cells in their microenvironment, including pericytes, for their proper function. Here, we show that Tcf7l2 activity in pancreatic pericytes is required for β-cell function. Transgenic mice in which Tcf7l2 was selectively inactivated in their pancreatic pericytes exhibited impaired glucose tolerance due to compromised β-cell function and glucose-stimulated insulin secretion. Inactivation of pericytic Tcf7l2 was associated with impaired expression of genes required for β-cell function and maturity in isolated islets. In addition, we identified Tcf7l2-dependent pericytic expression of secreted factors shown to promote β-cell function, including bone morphogenetic protein 4 (BMP4). Finally, we show that exogenous BMP4 is sufficient to rescue the impaired glucose-stimulated insulin secretion of transgenic mice, pointing to a potential mechanism through which pericytic Tcf7l2 activity affects β-cells. To conclude, we suggest that pancreatic pericytes produce secreted factors, including BMP4, in a Tcf7l2-dependent manner to support β-cell function. Our findings thus propose a potential cellular mechanism through which abnormal TCF7L2 activity predisposes individuals to diabetes and implicates abnormalities in the islet microenvironment in this disease. [ABSTRACT FROM AUTHOR]

Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia 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, P2Xcilia 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 P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high-strength field site. Ca2+ has been estimated to be 7-fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN-62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits. [ABSTRACT FROM AUTHOR]

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 γ2) 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 rules, 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. [ABSTRACT FROM AUTHOR]

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. Lesten 1999. J. Neurosci. 19:7289-7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transtected 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 multipie 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. [ABSTRACT FROM AUTHOR]

Snake and spider envenomation have a considerable impact on public health. Their pathology is induced by a variety of toxins composing the venom which induce cytotoxicity to cells of different organs by several cell death pathways. Described in this chapter are methods in vitro used to assess venoms and toxin-induced cell death using mammalian cell cultures. The chapter is divided into five sections: (1) a brief overview of in vitro cytotoxicity and categories of cell death induced by venoms and toxins; (2) a common method to measure necrotic cell death using lactate dehydrogenase (LDH) release; (3) a flow cytometry method that simultaneously measures necrosis and apoptosis; (4) measurements of nuclear morphology; and (5) measurements of the autophagy following microtubule-associated protein light chain 3 (LC3) expression, by immunoblotting and by fluorescence microscopy of LC3-positive vesicles, to assess the levels of autophagosomes.

Many toxins from a variety of venomous animals and plants have evolved to target neuronal ion channels and receptors. However, a significant obstacle in the study of these toxins is the finding and characterization of their specific molecular target. Here, we describe a method for fast and efficient screening of venom and toxin activity using live-cell calcium imaging. We describe the use of Fura-2, a calcium indictor that changes its fluorescence properties in response to intracellular calcium elevations, to measure the activity of neurons from the dorsal root and trigeminal ganglia. Calcium imaging is an efficient technique for testing many of the venom's components on large numbers of neurons simultaneously. This technique offers a novel tool for low-cost and rapid characterization of functionally active toxins and their target receptors.

Altered glucose reabsorption via 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 (CB 1 R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB 1 R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB 1 R blockade or genetically inactivating CB 1 Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB 1 R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB 1 R 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 CB 1 R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN., (Copyright © 2018 by the American Society of Nephrology.)