Endocrine disruptors in plastics alter ß-cell physiology and increase the risk of diabetes mellitus.

Plastic pollution breaks a planetary boundary threatening wildlife and humans through its physical and chemical effects. Of the latter, the release of endocrine disrupting chemicals (EDCs) has consequences on the prevalence of human diseases related to the endocrine system. Bisphenols (BPs) and phthalates are two groups of EDCs commonly found in plastics that migrate into the environment and make low-dose human exposure ubiquitous. Here we review epidemiological, animal, and cellular studies linking exposure to BPs and phthalates to altered glucose regulation, with emphasis on the role of pancreatic ß-cells. Epidemiological studies indicate that exposure to BPs and phthalates is associated with diabetes mellitus. Studies in animal models indicate that treatment with doses within the range of human exposure decreases insulin sensitivity and glucose tolerance, induces dyslipidemia, and modifies functional ß-cell mass and serum levels of insulin, leptin, and adiponectin. These studies reveal that disruption of ß-cell physiology by EDCs plays a key role in impairing glucose homeostasis by altering the mechanisms used by ß-cells to adapt to metabolic stress such as chronic nutrient excess. Studies at the cellular level demonstrate that BPs and phthalates modify the same biochemical pathways involved in adaptation to chronic excess fuel. These include changes in insulin biosynthesis and secretion, electrical activity, expression of key genes, and mitochondrial function. The data summarized here indicate that BPs and phthalates are important risk factors for diabetes mellitus and support a global effort to decrease plastic pollution and human exposure to EDCs.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERß in pancreatic beta cells.

17ß-estradiol protects pancreatic ß-cells from apoptosis via the estrogen receptors ERα, ERß and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERß, or in dispersed islet cells from ERß knockout (BERKO) mice. However, the ERα and ERß agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERß form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERß as well as GPER activation by G1 decreased ERαß heterodimers. We propose that ERαß heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαß heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis.

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3-Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin-Kv1.3 axis during cell survival and apoptosis.

Physiology of pancreatic ß-cells: Ion channels and molecular mechanisms implicated in stimulus-secretion coupling.

The human and mouse islet of Langerhans is an endocrine organ composed of five different cells types; insulin-secreting ß-cells, glucagon-producing α-cells, somatostatin-producing δ-cells, pancreatic polypeptide-secreting PP cells and ɛ-cells that secretes ghrelin. The most important cells are the pancreatic ß-cells that comprise around 45-50% of human islets and 75-80% in the mouse. Pancreatic ß-cells secrete insulin at high glucose concentration, thereby finely regulating glycaemia by the hypoglycaemic effects of this hormone. Different ion channels are implicated in the stimulus-secretion coupling of insulin. An increase in the intracellular ATP concentration leads to closure KATP channels, depolarizing the cell and opening voltage-gated calcium channels. The increase of intracellular calcium concentration induced by calcium entry through voltage-gated calcium channels promotes insulin secretion. Here, we briefly describe the diversity of ion channels present in pancreatic ß-cells and the different mechanisms that are responsible to induce insulin secretion in human and mouse cells. Moreover, we described the pathophysiology due to alterations in the physiology of the main ion channels present in pancreatic ß-cell and its implication to predispose metabolic disorders as type 2 diabetes mellitus.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in ß-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Bisphenol A Regulates Sodium Ramp Currents in Mouse Dorsal Root Ganglion Neurons and Increases Nociception.

17ß-Estradiol mediates the sensitivity to pain and is involved in sex differences in nociception. The widespread environmental disrupting chemical bisphenol A (BPA) has estrogenic activity, but its implications in pain are mostly unknown. Here we show that treatment of male mice with BPA (50 µg/kg/day) during 8 days, decreases the latency to pain behavior in response to heat, suggesting increased pain sensitivity. We demonstrate that incubation of dissociated dorsal root ganglia (DRG) nociceptors with 1 nM BPA increases the frequency of action potential firing. SCN9A encodes the voltage-gated sodium channel Nav1.7, which is present in DRG nociceptors and is essential in pain signaling. Nav1.7 and other voltage-gated sodium channels in mouse DRG are considered threshold channels because they produce ramp currents, amplifying small depolarizations and enhancing electrical activity. BPA increased Nav-mediated ramp currents elicited with slow depolarizations. Experiments using pharmacological tools as well as DRG from ERß-/- mice indicate that this BPA effect involves ERα and phosphoinositide 3-kinase. The mRNA expression and biophysical properties other than ramp currents of Nav channels, were unchanged by BPA. Our data suggest that BPA at environmentally relevant doses affects the ability to detect noxious stimuli and therefore should be considered when studying the etiology of pain conditions.

Oestrogen receptor ß mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells.

AIMS/HYPOTHESIS: Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and ß (ERß). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. METHODS: Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100 µg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor ß (ERß) knockout (Erß-/-) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. RESULTS: Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERß-/- mice did not present BPA-induced changes, suggesting that ERß mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. CONCLUSIONS/INTERPRETATION: Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERß in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.

Author Correction: First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

Among macrocyclic lactones (ML), ivermectin (IVM) and moxidectin (MOX) potentially affect all Ecdysozoan species, with dung beetles being particularly sensitive. The comparative effects of IVM and MOX on adult dung beetles were assessed for the first time to determine both the physiological sub-lethal symptoms and pre-lethal consequences. Inhibition of antennal response and ataxia were tested as two intuitive and ecologically relevant parameters by obtaining the lowest observed effect concentration (LOEC) values and interpolating other relevant toxicity thresholds derived from concentration-response curves (IC50, as the concentration of each ML where the antennal response is inhibited by half; and pLC50, as the quantity of ingested ML where partial paralysis was observed by half of treated individuals) from concentration-response curves. Both sub-lethal and pre-lethal symptoms obtained in this study coincided in that IVM was six times more toxic than MOX for adult dung beetles. Values of LOEC, IC50 and pLC50 obtained for IVM and MOX evaluated in an environmental context indicate that MOX, despite needing more time for tis elimination in the faeces, would be twice as harmful to dung beetles as IVM. This approach will be valuable to clarify the real impact of MLs on dung beetle health and to avoid the subsequent environmental consequences.

A Calcium-Dependent Chloride Current Increases Repetitive Firing in Mouse Sympathetic Neurons.

Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9'-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.

Author Correction: Molecular mechanisms involved in the non-monotonic effect of bisphenol-a on Ca2+ entry in mouse pancreatic ß-cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Extranuclear-initiated estrogenic actions of endocrine disrupting chemicals: Is there toxicology beyond paracelsus?

Endocrine Disrupting Chemicals (EDCs), including bisphenol-A (BPA) do not act as traditional toxic chemicals inducing massive cell damage or death in an unspecific manner. EDCs can work upon binding to hormone receptors, acting as agonists, antagonists or modulators. Bisphenol-A displays estrogenic activity and, for many years it has been classified as a weak estrogen, based on the classic transcriptional action of estrogen receptors serving as transcription factors. However, during the last two decades our knowledge about estrogen signaling has advanced considerably. It is now accepted that estrogen receptors ERα and ERß activate signaling pathways outside the nucleus which may or may not involve transcription. In addition, a new membrane estrogen receptor, GPER, has been proposed. Pharmacological and molecular evidence, along with results obtained in genetically modified mice, demonstrated that BPA, and its substitute BPS, are potent estrogens acting at nanomolar concentrations via extranuclear ERα, ERß, and GPER. The different signaling pathways activated by BPA and BPS explain the well-known estrogenic effects of low doses of EDCs as well as non-monotonic dose-response relationships. These signaling pathways may help to explain the actions of EDCs with estrogenic activity in the etiology of different pathologies, including type-2 diabetes and obesity.

Molecular mechanisms involved in the non-monotonic effect of bisphenol-a on ca2+ entry in mouse pancreatic ß-cells.

In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma "the dose makes the poison". Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic ß-cells from wild-type and oestrogen receptor ERß-/- mice, we found that exposure to increasing doses of BPA affected Ca2+ entry in an NMDR manner. Low doses decreased plasma membrane Ca2+ currents after downregulation of Cav2.3 ion channel expression, in a process involving ERß. High doses decreased Ca2+ currents through an ERß-mediated mechanism and simultaneously increased Ca2+ currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca2+ entry in ß-cells.

Effects of Bisphenol A on ion channels: Experimental evidence and molecular mechanisms.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) produced in huge quantities in the manufacture of polycarbonate plastics and epoxy resins. It is present in most humans in developed countries, acting as a xenoestrogen and it is considered an environmental risk factor associated to several diseases. Among the whole array of identified mechanisms by which BPA can interfere with physiological processes in living organisms, changes on ion channel activity is one of the most poorly understood. There is still little evidence about BPA regulation of ion channel expression and function. However, this information is key to understand how BPA disrupts excitable and non-excitable cells, including neurons, endocrine cells and muscle cells. This report is the result of a comprehensive literature review on the effects of BPA on ion channels. We conclude that there is evidence to say that these important molecules may be key end-points for EDCs acting as xenoestrogens. However, more research on channel-mediated BPA effects is needed. Particularly, mechanistic studies to unravel the pathophysiological actions of BPA on ion channels at environmentally relevant doses.

Low doses of ivermectin cause sensory and locomotor disorders in dung beetles.

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin's effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes.

Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.

Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors in Xenopus oocytes.

Lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) play important roles in a variety of physiological and pathophysiological processes, including cell proliferation, angiogenesis, central nervous system development and carcinogenesis. Whilst many ion channels and transporters are recognized to be controlled by a change in cell membrane potential, little is known about the voltage dependence of other proteins involved in cell signalling. Here, we show that the InsP(3)-mediated Ca(2+) response stimulated by the endogenous LPA GPCR in Xenopus oocytes is potentiated by membrane depolarization. Depolarization was able to repetitively stimulate transient [Ca(2+)](i) increases after the initial agonist-evoked response. In addition, the initial rate and amplitude of the LPA-dependent Ca(2+) response were significantly modulated by the steady holding potential over the physiological range, such that the response to LPA was potentiated at depolarized potentials and inhibited at hyperpolarized potentials. Enhancement of LPA receptor-evoked Ca(2+) mobilization by membrane depolarization was observed over a wide range of agonist concentrations. Importantly, the amplitude of the depolarization-evoked intracellular Ca(2+) increase displayed an inverse relationship with agonist concentration such that the greatest effect of voltage was observed at near-threshold levels of agonist. Voltage-dependent Ca(2+) release was not induced by direct elevation of InsP(3) or by activation of heterotrimeric G-proteins in the absence of agonist, indicating that the LPA GPCR itself represents the primary site of action of membrane voltage. This novel modulation of LPA signalling by membrane potential may have important consequences for control of Ca(2+) signals both in excitable and non-excitable tissues.

A role for membrane potential in regulating GPCRs?

G-protein-coupled receptors (GPCRs) have ubiquitous roles in transducing extracellular signals into cellular responses. Therefore, the concept that members of this superfamily of surface proteins are directly modulated by changes in membrane voltage could have widespread consequences for cell signalling. Although several studies have indicated that GPCRs can be voltage dependent, particularly P2Y(1) receptors in the non-excitable megakaryocyte, the evidence has been mostly indirect. Recent work on muscarinic receptors has stimulated substantial interest in this field by reporting the first voltage-dependent charge movements for a GPCR. An underlying mechanism is proposed whereby a voltage-induced conformational change in the receptor alters its ability to couple to the G protein and thereby influences its affinity for an agonist. We discuss the strength of the evidence behind this hypothesis and include suggestions for future work. We also describe other examples in which direct voltage control of GPCRs can account for effects of membrane potential on downstream signals and highlight the possible physiological consequences of this phenomenon.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Potentiation of P2Y receptors by physiological elevations of extracellular K+ via a mechanism independent of Ca2+ influx.

Many physiological and pathophysiological situations generate a significant increase in extracellular K+ concentration. This is known to influence a number of membrane conductances and exchangers, whereas direct effects of K+ on the activation of G protein-coupled receptors have not been reported. We now show that Ca2+ release evoked by P2Y1 receptors expressed in 1321-N1 astrocytoma cells is markedly potentiated by small increases in external K+ concentration. This effect was blocked by the phospholipase-C inhibitor U-73122 (1-[6-[[17 beta]-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not by its analog U-73343 (1-[6-[[17 beta]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), and not by nifedipine, Ni2+, Cd2+, or Gd3+. Thus, K+ enhances d-myo-inositol 1,4,5-trisphosphate-dependent Ca2+ release without a requirement for Ca2+ influx. The cation dependence of this effect displayed the order K+ > Rb+ > N-methyl-D-glucamine+, and Cs+ and choline+ were ineffective. The potentiation by K+ is half-maximal at an increase of 2.6 mM (total K+ of 7.6 mM). K+ caused a reduction in EC50 (2.7-fold for a 29 mM increase) without a change of slope; thus, the greatest effect was observed at near-threshold agonist levels. The response to K+ can be explained in part by depolarization-dependent potentiation of P2Y1 receptors [J Physiol (Lond) 555:61-70, 2004]. However, electrophysiological recordings of 1321-N1 cells and megakaryocytes demonstrated that K+ also amplifies ADP-evoked Ca2+ responses independently of changes in membrane potential. Elevated K+ also amplified endogenous UTP-dependent Ca2+ responses in human embryonic kidney 293 cells, suggesting that other P2Y receptors are K(+)-dependent. P2Y receptors display a widespread tissue distribution; therefore, their modulation by small changes in extracellular K+ may represent a novel means of autocrine and paracrine regulation of cellular activity.