Search

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 degrees C) resulted in a more sustained depolarization-evoked Ca2+ increase compared with more transient or oscillatory responses at 20-24 degrees 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.

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.

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 P2Y1 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]