Juan Martinez-Pinna

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.

Properties of the demarcation membrane system in living rat megakaryocytes.

The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracellular indicator displayed the extensive nature of the channels formed by the DMS throughout the extranuclear volume. Under whole-cell patch clamp, the DMS is electrophysiologically contiguous with the peripheral plasma membrane such that a single capacitative component can account for the biophysical properties of all surface-connected membranes in the majority of recordings. Megakaryocyte capacitances were in the range of 64-694 pF, equivalent to 500-5500 platelets (mean value 1850). Based upon calculations for a spherical geometry, the DMS results in a 4- to 14-fold (average 8.1-fold) increase in specific membrane capacitance expressed per unit spherical surface area. This indicates a level of plasma membrane invagination comparable with mammalian skeletal muscle. Whole-cell capacitance measurements and confocal imaging of membrane-impermeant fluorescent indicators therefore represent novel approaches to monitor the DMS during megakaryocytopoiesis and thrombopoiesis.

Direct Voltage Control of Signaling via P2Y1 and Other Gαq-coupled Receptors

Emerging evidence suggests that Ca2+ release evoked by certain G-protein-coupled receptors can be voltage-dependent; however, the relative contribution of different components of the signaling cascade to this response remains unclear. Using the electrically inexcitable megakaryocyte as a model system, we demonstrate that inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization stimulated by several agonists acting via Gαq-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP. Voltage-dependent Ca2+ release was not induced by direct elevation of inositol 1,4,5-trisphosphate, by agents mimicking diacylglycerol actions, or by activation of phospholipase Cγ-coupled receptors. The response to voltage did not require voltage-gated Ca2+ channels as it persisted in the presence of nifedipine and was only weakly affected by the holding potential. Strong predepolarizations failed to affect the voltage-dependent Ca2+ increase; thus, an alteration of G-protein βγ subunit binding is also not involved. Megakaryocytes from P2Y1-/- mice lacked voltage-dependent Ca2+ release during the application of ADP but retained this response after stimulation of other Gαq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPγS dialysis and to a lesser extent during \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{AlF}_{4}^{-}\) \end{document} or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Gαq-coupled receptors is not due to control of G-proteins or down-stream signals but, rather, can be explained by a voltage sensitivity at the level of the receptor itself. This effect, which is particularly robust for P2Y1 receptors, has wide-spread implications for cell signaling.

Distinct mechanisms for activation of Cl− and K+ currents by Ca2+ from different sources in mouse sympathetic neurones

1 We have investigated the roles of different voltage‐dependent Ca2+ channels in the activation of the Cl− and K+ channels responsible for the afterdepolarization (ADP) and slow afterhyperpolarization (AHP) in sympathetic neurones of the isolated mouse superior cervical ganglion in vitro. 2 The ADP and its associated Ca2+‐activated Cl− current were markedly decreased by ω‐agatoxin IVA (40–200 nm) and nifedipine (1–10 μm), but not by ω‐conotoxin GVIA (300 nm). 3 In contrast, the AHP and the apamin‐sensitive Ca2+‐activated K+ current that underlies this potential were blocked by ω‐conotoxin GVIA, but were not affected by ω‐agatoxin IVA and were only slightly reduced by nifedipine. 4 Ryanodine (20 μm) reduced the Ca2+‐activated Cl− current following an action potential by 75 % but on average did not affect the Ca2+‐activated K+ current. 5 Evidence that R‐type channels provide a proportion of the Ca2+ activating both types of Ca2+‐dependent channel was obtained. 6 We conclude that Ca2+ entering through L‐ and P‐type Ca2+ channels preferentially activates the Cl− current responsible for the ADP in mouse sympathetic neurones, predominantly via Ca2+‐induced Ca2+ release, whereas the Ca2+ that activates the K+ channels responsible for the AHP enters predominantly through N‐type channels. The data can be explained by the selective association of each type of Ca2+ channel with particular intracellular mechanisms for activating other membrane channels, one indirect and the other direct, probably located at discrete sites on the soma and dendrites.

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.

Sensitivity limits for voltage control of P2y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G‐protein‐coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non‐excitable rat megakaryocyte as a model system, we now show that P2Y receptor‐evoked Ca2+ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca2+ concentration ([Ca2+]i) in response to depolarization was always larger than the maximal reduction in [Ca2+]i following an equivalent amplitude hyperpolarization. Significant [Ca2+]i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor‐evoked Ca2+ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca2+]i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization‐evoked Ca2+ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G‐protein‐coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non‐excitable tissues.

Calcium current components in intact and dissociated adult mouse sympathetic neurons

We examined which types of high threshold Ca(2+) channels are activated by depolarization in intact and dissociated sympathetic neurons from adult mouse superior cervical ganglia (SCG). Ba(2+) currents were recorded with microelectrodes and discontinuous voltage clamp from neurons in intact ganglia, and using the perforated patch clamp technique in dissociated cells. Peak current was larger in intact neurons, although the voltage dependence was similar. Successive application of omega-conotoxin GVIA, omega-conotoxin MVIIC and nifedipine revealed that the total current in intact cells was composed by 29% N-type, 13% P/Q-type, 32% L-type and 26% resistant to blockade (R-type). In dissociated cells, the N component was larger and the L component smaller, whereas P/Q-type and R-type were similar. Peak currents evoked with an action potential waveform instead of a square pulse were larger in both preparations but the proportions of each component were similar. We conclude that dissociating and culturing somata results in data that only partially reflect the situation in intact neurons. Assuming that the main effect of dissociation is the removal of mature dendritic membrane, the data suggest that L channels are more abundant on dendrites and N channels on the soma of intact sympathetic neurons, whereas P/Q and R channels may be uniformly distributed over the cell surface. Finally, in intact SCG neurons from rats, the proportions of current evoked by a pulse were: 49% N-type, 11% P/Q-type, 21% L-type and 20% R-type when nifedipine was applied last, suggesting that there are species differences in the expression of L and N channels.

Novel consequences of voltage‐dependence to G‐protein‐coupled P2Y1 receptors

Emerging evidence suggests that activation of G‐protein‐coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non‐excitable megakaryocyte using a range of agonists and antagonists.

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 ERβ 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, ERβ, 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.

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

J. Neurochem. (2011) 117, 1009–1019.