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Originally published In Press as doi:10.1074/jbc.M407783200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 2, 1490-1498, January 14, 2005
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Direct Voltage Control of Signaling via P2Y1 and Other G{alpha}q-coupled Receptors*

Juan Martinez-Pinna{ddagger}§, Iman S. Gurung{ddagger}§, Catherine Vial||, Catherine Leon**, Christian Gachet**, Richard J. Evans||, and Martyn P. Mahaut-Smith{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom, ||Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom, and **INSERM U.311, EFS-Alsace 10, Strasbourg Cedex, 67065, France

Received for publication, July 12, 2004 , and in revised form, November 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 G{alpha}q-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 C{gamma}-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 {beta}{gamma} 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 G{alpha}q-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTP{gamma}S dialysis and to a lesser extent during or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via G{alpha}q-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G-protein-coupled receptors (GPCRs)1 constitute the largest family of surface proteins and represent key targets for therapeutic intervention (1, 2). Although GPCR activation is not normally considered to be directly sensitive to changes in the cell membrane potential, studies in a variety of cell types now support this concept (3–7). In particular, Ca2+ release stimulated by P2Y receptors in rat megakaryocytes or muscarinic cholinergic receptors in coronary artery smooth muscle is potentiated by depolarization and inhibited by hyperpolarization (5, 7, 8). The underlying mechanism is unknown and may lie at the level of the receptor, a downstream signaling molecule, or reflect a direct effect on the intracellular Ca2+ stores.

Voltage control of GPCR signaling represents a potentially important means whereby electrogenic influences can modify cellular signaling. Indeed, a range of physiological voltage waveforms from slow oscillations to action potentials can alter the Ca2+ mobilization induced by P2Y receptor activation (8, 9). Constitutive voltage control of phospholipase C and, thus, IP3-dependent Ca2+ release has also been described in smooth and skeletal muscle (10, 11), inferring that activation of heterotrimeric G-proteins or their receptors can be voltage-dependent in the absence of exogenous agonist. To date, the voltage dependence to GPCR signaling has been most extensively studied in the rat megakaryocyte, where the lack of voltage-gated Ca2+ influx and ryanodine receptors simplifies studies of IP3-dependent Ca2+ mobilization (12–14). In addition, due to its role in generating functional anucleated blood platelets, the megakaryocyte cell surface possesses a plethora of platelet surface receptors with relatively well defined signaling pathways (15). We have now used the megakaryocyte, including tissues from receptor-deficient mice, to investigate the location of the voltage sensor within the signaling cascade coupled to P2Y and other G{alpha}q-coupled receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Isolation and Animals—Marrow was dissociated from the femoral and tibial bones of male adult C57BL/6 mice and Wistar rats into standard external saline (see "Solutions and Reagents") containing 0.32 units ml-1 type V or type VII apyrase. Apyrase was present throughout storage but omitted during experiments except where indicated. Rat marrow provides a higher yield of megakaryocytes compared with the mouse and, thus, was used for many experiments due to the rarity (<1%) of these cells. Megakaryocytes were distinguished on the basis of their large size (16), and recordings were made within 12 h of marrow removal. P2Y1 receptor-deficient (P2Y1-/-) mice were generated as previously described (17).

Solutions and Reagents—The standard external saline contained 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM D-glucose titrated to pH 7.35 with NaOH. Application of extracellular solutions was by gravity-driven bath superfusion. For Ca2+-free saline, CaCl2 was replaced by an equal concentration of MgCl2. For Na+-free saline, Na+ was replaced by an equal concentration of n-methyl-D-glucamine, and the pH was titrated to 7.35 with n-methyl-D-glucamine. The pipette saline contained 150 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.05 mM Na2GTP, 10 mM Hepes, pH 7.2 (adjusted with KOH), and either 0.05 mM K5fura-2 or 0.05 mM (NH4)5fluo-3. For experiments with internal GTP{gamma}S (tetralithium salt; Sigma-Aldrich), Na2GTP was omitted from the pipette saline. K5fura-2 and (NH4)5fluo-3 were purchased from Molecular Probes (Eugene, OR). ADP (Sigma) was pretreated for 1 h with hexokinase and high glucose to remove contaminating levels of ATP, which co-activates P2X receptors, as described previously (18). Caged-IP3, caged-G-PIP2 and 1-oleoyl-2-acetylglycerol (OAG) were obtained from (Calbiochem-Novabiochem). Platelet-derived growth factor-BB (PDGF) and phorbol 12-myristate 13-acetate were purchased from Sigma. Cells were exposed to by the addition of 5 mM NaF and 100 µM AlCl3 to the normal external saline. For assessment of the relative voltage dependence to different receptors, we selected agonist concentrations that generated a robust Ca2+ response and which were greater than the reported EC50 value in the platelet and/or megakaryocyte (19–22). This will limit concentration-dependent effects for the depolarization-evoked increase, which varies by only 0.74-fold over a 100-fold concentration range above the EC50 value.2

Electrophysiology—Conventional whole-cell patch clamp recordings were carried out in voltage clamp mode using an Axopatch 200B amplifier (Axon Instruments, CA) with 70–75% series resistance compensation. pCLAMP and a Digidata interface (Axon instruments) were used to deliver voltage steps. Voltage-dependent Ca2+ mobilization was assessed using 80-mV depolarizations of 2–10-s duration, which stimulate a near-maximal voltage-dependent [Ca2+]i increase during exposure to the agonist ADP (9). For the experiments shown in Fig. 3C, the specified holding potential was applied before ADP, and the effect of the 80-mV depolarization was assessed as soon as possible after the initial agonist-evoked [Ca2+]i transients. Although this approach may have introduced some variation in internal Ca2+ store content as a consequence of different driving forces for Ca2+ influx, it avoided the voltage-dependent Ca2+ responses caused by changing the holding potential during activation of the G-protein-coupled receptor. Recordings were made at room temperature (20–24 °C) for improved stability of recordings, although we have previously confirmed that voltage control of P2Y receptor-evoked Ca2+ release also occurs at normal body temperatures (9).



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  		FIG. 3.
Potentiation of P2Y1 receptor Ca2+ responses is maintained in the presence of nifedipine and is weakly dependent upon holding potential. A, representative trace showing the ability of depolarization (80 mV, 10-s duration) to potentiate ADP-evoked Ca2+ mobilization in the presence of nifedipine (50 µM). The whole-cell current is also shown in A (middle trace) to demonstrate the inhibition of voltage-gated K+ current by nifedipine. B, average peak [Ca2+]i increases (5–17 cells, as indicated) evoked by 80-mV, 10-s duration depolarizations in the absence of agonist, in the presence of 1 µM ADP, or in the presence of both 1 µM ADP and 50 µM nifedipine. Nifedipine was applied for either 3–5 min (Nif (short)) or >20 min (Nif (long)) before depolarization. C, average peak [Ca2+]i increases evoked by 80-mV, 10-s-duration depolarizations from different holding potentials (Vhold; n = 5–19 cells for each point). Cells were held at each holding potential throughout application of agonist and depolarization.

 
Fluorescence Measurements—Fura-2 or fluo-3 fluorescence measurements of intracellular Ca2+ were made using a Cairn Spectrophotometer System (Cairn Research Ltd, Kent, UK) coupled to a Nikon Diaphot inverted microscope (Nikon, Japan), as described in detail elsewhere (23, 24). Excitation wavelengths, provided by a monochromator or using interference filters, were 340 and 380 nm for fura-2 and 490 nm for fluo-3 with emission bandwidths of ~480–600 and 528–600 nm, respectively. Fluorescence emission and electrophysiological data were simultaneously recorded to computer using Cairn acquisition hardware and software. Fluorescence data were sampled at 60 Hz, averaged to give a final acquisition rate of 15 Hz, and exported for analysis within Microcal Origin (Microcal Software Inc., Northampton, MA). For calibration of fura-2, the maximum and minimum 340/380-nm fluorescence ratios were measured extracellularly, and background-corrected ratios were converted to [Ca2+]i as described previously (25) after application of a viscosity correction factor of 0.85 (23, 26). A Kd of 258 nM was derived using a calibration kit from Molecular Probes. Data are expressed as the means ± S.E. with the statistical difference assessed using Student's unpaired t test. Fluo-3 fluorescent signals were background-subtracted, and f/f0 ratios were used to normalize fluorescence levels (f) against starting fluorescence (f0). Data are expressed as the means ± S.E. of the mean, where n is the number of cells. Statistical comparisons were made using Student's unpaired t test, and p < 0.05 was considered significant.

Flash Photolysis of Caged Compounds—Cells were loaded with caged-IP3 (100 µM) or caged-G-PIP2 (200 µM) by dialysis from the patch pipette. Uncaging was achieved using a Cairn Flash Photolysis unit (Cairn Research Ltd, Faversham, Kent, UK) with the amount of photolysis determined by the charge delivered to a high intensity xenon arc lamp (Advance Radiation, Inc., Santa Clara, CA). A 503-nm dichroic mirror with extended UV reflectance allowed simultaneous delivery of uncaging and 490-nm illumination as described in detail elsewhere (12, 23).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Voltage-dependent Ca2+ Release during Activation of Multiple G{alpha}q-coupled GPCRs—The megakaryocyte is a useful cell type for studying voltage control of GPCR-evoked Ca2+ signaling as this electrically inexcitable cell type expresses multiple surface receptors known to couple to Ca2+ mobilization in the platelet. Consequently, the platelet agonists ADP, U46619 [GenBank] (a stable thromboxane A2 analogue), and 5-HT, which stimulate phospholipase C{beta} via G{alpha}q-coupled receptors (27–30), all evoked a robust elevation of intracellular Ca2+ concentration ([Ca2+]i) in the rat megakaryocyte (Fig. 1, A–C). In the absence of exogenous agonist, depolarization had little or no effect on [Ca2+]i (Fig. 1, A–E). However, despite the lack of functional voltage-gated Ca2+ channels (7, 13, 31), depolarization from -75 to +5 mV repeatedly stimulated a significant [Ca2+]i increase during stimulation by each of the three agonists tested.



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  		FIG. 1.
Comparison of agonist- and voltage-dependent Ca2+ mobilization during activation of different G{alpha}q-coupled receptors. Simultaneous [Ca2+]i and whole-cell patch clamp (voltage-clamp) recordings from rat megakaryocytes. The effect of 10-s-duration depolarizing steps from -75 to 5 mV was tested in the presence and absence of 1 µM ADP (A and D), 300 nM U46619 [GenBank] (B), and 10 µM 5-HT (C). E, average peak [Ca2+]i increases evoked by each agonist alone and by depolarization in the presence or absence (first column) of the agonist. The number of cells is indicated below each group. Experiments in A, B, C, and E were conducted in the presence of normal external Ca2+ (1 mM); in D, the agonist was applied in Ca2+-free saline with Mg2+ added to maintain a constant divalent cation concentration.

 
The agonist-induced depolarization-evoked Ca2+ increase results primarily from release of intracellularly stored Ca2+,asit was of similar amplitude in Ca2+-free medium (see Fig. 1D for a typical experiment with ADP; data are not shown for thromboxane A2 (n = 9) and 5-HT (n = 5)). However, depletion of stores frequently led to a run-down of the response; therefore, subsequent experiments were conducted in the presence of external Ca2+. Furthermore, although voltage changes are capable of modulating the initial agonist-evoked Ca2+ signals, including oscillations (8, 32), depolarizations in this study were applied during the steady plateau phase of the response to clearly distinguish voltage-dependent effects. Fig. 1E compares the peak agonist and depolarization-evoked Ca2+ increases in the presence of external Ca2+. The rank order of ability for depolarization to potentiate Ca2+ mobilization during agonist stimulation was ADP > U46619 [GenBank] > 5-HT, measured both as the absolute voltage-dependent Ca2+ increase (315 ± 24, 118 ± 17, and 52 ± 14 nM for ADP (n = 19), U46619 [GenBank] (n = 18), and 5-HT (n = 13), respectively) and the percentage Ca2+ increase for depolarization compared with that evoked by the agonist (52 ± 5, 33 ± 6, and 11 ± 4% for ADP, U46619 [GenBank] , and 5-HT, respectively). Thus, several G{alpha}q-coupled receptors are able to induce the ability of membrane potential to control Ca2+ mobilization, although this response is most pronounced for ADP-stimulated P2Y receptors.

Lack of Voltage-dependent Ca2+ Release after Direct Elevation of IP3 and/or Diacylglycerol Analogues and Phospholipase C{gamma} Activation—The voltage dependence of Ca2+ release during activation of a GPCR can be explained by direct regulation of the receptor, its G-protein, or phospholipase C by the cell membrane potential (5, 7, 12, 33). To assess whether the products of phospholipase C can alone induce a voltage dependence to Ca2+ release, for example by configurational coupling between IP3 receptors on the internal stores and ion channels on the plasma membrane (34), we tested the effects of elevating IP3 with and without stimulation of diacylglycerol (DAG)-dependent pathways. To limit the effect of reduced Ca2+ influx when the cell is depolarized, the duration of the voltage step was shortened to 2s, which we have recently shown maximally activates the voltage-dependent Ca2+ release process (9). Depolarizing voltage steps could not stimulate a Ca2+ increase after uncaging of IP3 by flash photolysis (n = 9; Fig. 2A). The lack of response to voltage was not due to complete inactivation of IP3 receptors or rapid degradation of IP3 since depolarization also failed to stimulate Ca2+ increase after photolysis of the poorly metabolizable but fully active IP3 analogue, G-PIP2, which itself could repeatedly release Ca2+ (n = 10; Fig. 2B) (35, 36). We also tested the effects of depolarization during application of the cell-permeant DAG analogue, OAG (20 µM, n = 5; 100 µM, n = 9). The Ca2+ response to this compound was variable at either concentration, ranging from no increase (3/14 cells) to a small (≤150 nM) increase (11/14 cells), which may in part be due to stimulation of Ca2+ entry via TRPC channels (canonical members of the transient receptor potential family of ion channels) (37, 38). However, in all cells, depolarization of the membrane (from -75 to +5 mV, 10 s duration) failed to produce a Ca2+ increase (not shown). We also activated the DAG target protein kinase-C using phorbol 12-myristate 13-acetate at 200 nM (n = 6) or 1 µM (n = 10). As with OAG, phorbol 12-myristate 13-acetate evoked a Ca2+ increase in a proportion of cells (8/16); however, in all cells (n = 16), depolarization from -75 to +5 mV failed to stimulate a Ca2+ increase (not shown). Finally, elevation of both IP3, by flash photolysis combined with either 1 µM phorbol 12-myristate 13-acetate (n = 4, not shown) or 100 µM OAG (n = 8; Fig. 2C) failed to induce voltage-dependent Ca2+ release. Together, these experiments exclude the possibility that the voltage dependence during GPCR activation results from an event induced by direct activation of IP3 receptors or DAG-dependent pathways.



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  		FIG. 2.
Membrane depolarization fails to stimulate Ca2+ mobilization during direct elevation of phospholipase C products or after receptor-dependent activation of phospholipase C{gamma}. [Ca2+]i recordings (fluo-3 f/f0 ratio) during photolytic release of IP3 (A), G-PIP2 (B), or IP3 in the presence of 100 µM OAG (C) are shown. The cell membrane potential was clamped at either -75 or 5 mV as indicated by the lower panels. A and C show two traces from different sections of the same experiment in which the potential was -75 mV throughout (black traces) or was stepped to 5 mV for 2 s after the uncaging event (red). Arrows indicate application of a brief ({approx}2 ms) flash of UV illumination at a constant intensity for each experiment. D, stimulation of phospho-lipase C{gamma}-coupled PDGF receptors fails to induce voltage-dependent Ca2+ release.

 
Platelets and megakaryocytes also express receptors coupled to Ca2+ mobilization via phospholipase C{gamma}, including those for platelet-derived growth factor (PDGF) (39). This agonist was slow to generate a Ca2+ increase, as expected at room temperature for this type of tyrosine-kinase-linked pathway, however no depolarization-induced response was observed leading up to or during the initial PDGF-induced Ca2+ increase (n = 5, Fig. 2D). Again, this supports a location for the voltage sensor upstream of phosphatidylinositol 4,5-bisphosphate hydrolysis or IP3 and DAG-dependent events.

Dihydropyridine Receptors and Other Voltage-gated Ca2+ Channels Are Not Involved in the Voltage Dependence to G{alpha}q Protein-coupled Receptors—Depolarization-evoked IP3-dependent Ca2+ release has been reported in skeletal and vascular smooth muscle, where dihydropyridine receptors coupled either directly or indirectly to G-proteins act as the voltage sensors (10, 11). Exogenous agonist is not required for the response; however, constitutive GPCR activity may still be involved. To test whether modified voltage-dependent Ca2+ channels are involved in the megakaryocyte voltage-gated Ca2+ release phenomenon, we examined the effects of a dihydropyridine and different holding potentials. Nifedipine (up to 50 µM), a blocker of L-type Ca2+ channels and myocyte voltage-gated Ca2+ release, had no significant effect on the ability of depolarization to potentiate P2Y1 receptors during activation by 1 µM ADP in the rat megakaryocyte (Fig. 3, A and C). A high concentration of nifedipine was used because it inhibited the voltage-gated K+ currents, thereby acting as a positive control (see Fig. 3A). Neither short term (2–5 min) nor long term (>20 min) exposures to nifedipine significantly affected the peak of the voltage-dependent Ca2+ increase (Fig. 3B).

ADP-induced Ca2+ mobilization was potentiated by a depolarizing step (80 mV, 10-s duration) applied from a wide range of holding potentials, although the peak response gradually declined as the holding potential was shifted from -102.5 to +7.5 mV (Fig. 3C). This may in part result from the reduced driving force for Ca2+ entry and, thus, a lower level of Ca2+ in the stores as the cell is held more depolarized (see "Experimental Procedures"). However, it does demonstrate the ability of membrane voltage to release a significant amount of Ca2+ from internal stores at holding potentials of -20 mV and more depolarized. This further argues against the role of voltage-gated Ca2+ ion channels in the underlying mechanism, since these are largely inactive over this range of holding potentials (40). The series of experiments in Fig. 3C also demonstrates that the response to voltage is highly robust over the range of normal resting potentials, which fits with postulated roles for this phenomenon, namely control of agonist-evoked Ca2+ oscillations and synergy between post-synaptic potentials or action potentials and GPCRs (8, 9).

One well established, weakly voltage-dependent process during GPCR activation is the inhibitory action of G-protein {beta}{gamma} subunits on voltage-gated Ca2+ channels (41–43). We, therefore, tested a predepolarization protocol (175mV, 15-ms depolarization from -75 mV) that mimics this effect in neurons but was too brief to cause voltage-dependent Ca2+ release in the megakaryocyte. Application of this strong predepolarization had no significant effect on the voltage-dependent Ca2+ increase evoked by a 4-s, 80-mV depolarization from -75 mV; the average peak Ca2+ increase was 228 ± 19 nM for control steps versus 212 ± 30 nM for steps applied 4.5 ms after the predepolarization; n = 5, p > 0.05). Overall, therefore, these data argue against a role for dihydropyridine receptors or other voltage-gated Ca2+ channels or shifts of G-protein {beta}{gamma} subunit binding in the voltage sensor responsible for controlling Ca2+ signaling via GPCRs.

Requirement of P2Y1 Receptors for the Voltage-dependent Ca2+ Release Induced by ADP and Thimerosal but Not Other G{alpha}q-coupled Receptors—ADP is known to stimulate platelets via two G-protein-coupled receptors, P2Y1 and P2Y12, which interact synergistically to generate full platelet aggregation responses (44–46). ADP-evoked Ca2+ mobilization in the platelet has been reported to be due either entirely to P2Y1 receptors (17, 47, 48) or to also involve P2Y12 receptors (49–51). We, therefore, turned to P2Y1 receptor-deficient (P2Y1-/-) mice to assess the relative role of different P2Y receptors in the ADP-dependent depolarization-evoked Ca2+ response in the megakaryocyte. ADP induced a Ca2+ increase (344 ± 47 nM, n = 26) and a robust voltage-dependent Ca2+ increase (232 ± 48 nM, n = 26) in wild type mouse megakaryocytes (Fig. 4A). As shown previously for rat megakaryocytes (7), this effect of membrane potential is predominantly due to release of Ca2+ from internal stores as it is observed in Ca2+-free (Fig 4B; n = 7) and Na+-free medium (n = 12, data not shown). P2Y1 receptors were required for the Ca2+ responses to both ADP (1 µM) and depolarization as they were absent in P2Y1-/- megakaryocytes (43 of 43 cells; see Fig. 4C). Depolarization-evoked Ca2+ increases were still observed during exposure to U46619 [GenBank] and 5-HT in P2Y1-/- megakaryocytes (Fig. 5, A and B; n = 15 and 19, respectively); thus, functional G{alpha}q-coupled receptor responses remain intact in this genomic model. We have previously demonstrated that the thiol reagent thimerosal, which elevates [Ca2+]i via both sensitization of IP3 receptors and depletion of the internal stores (52), can induce a small voltage-dependent Ca2+ release in rat megakaryocytes (12). This response was also observed in the mouse megakaryocyte (Fig. 5C, n = 7) and can be attributed to the action of thimerosal on sulfhydryl groups, as it was reversed by the reducing agent dithiothreitol (Fig. 5C) (52). However depolarization-evoked Ca2+ release was not observed in P2Y1-/- megakaryocytes during the response to thimerosal (Fig. 5D; n = 19), indicating that stimulation of P2Y1 receptors, for example via constitutive or autocrine activation (53, 54), is necessary for the ability of thimerosal to induce the voltage-dependent response rather than via the sensitization of IP3 receptors per se. Together with the lack of response to depolarization during stimulation of PDGF receptors or release of phospholipase C products (see earlier), this confirms that the principal voltage sensor is upstream of IP3 production.



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  		FIG. 4.
P2Y1 receptors are essential for induction of voltage-dependent Ca2+ release by ADP. [Ca2+]i responses of wild type (WT; A and B) and P2Y1-/- mouse megakaryocytes (C) to 1 µM ADP and 10-s step depolarizations from -75 to + 5 mV in standard external saline (A and C) or Ca2+-free medium (B).

 



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  		FIG. 5.
Megakaryocytes lacking P2Y1 receptors retain voltage-dependent Ca2+ responses during activation of other G{alpha}q-coupled receptors but not after IP3 receptor sensitization. [Ca2+]i responses of mouse P2Y1-/- megakaryocytes to 300 nM U46619 [GenBank] (A) and 10 µM 5-HT (B) and step depolarizations from a holding potential of -75 to + 5 mV. Sensitization of IP3 receptors by 100 µM thimerosal (thim) induced a small voltage-dependent Ca2+ release in wild type (WT) mouse megakaryocytes (C). The effects of thimerosal were reversed by 2 mM dithiothreitol (DTT). Thimerosal induced a [Ca2+]i increase in P2Y1-/- megakaryocytes, but voltage steps were unable to generate a [Ca2+]i increase (D).

 
Effect of Heterotrimeric G-protein Activation by Intracellular GTP{gamma}S or Aluminum Tetrafluoride—Intracellular application of the poorly hydrolysable GTP analogue, GTP{gamma}S, is frequently employed as a tool to activate heterotrimeric G-proteins in the absence of agonist (55). In wild type murine megakaryocytes, the addition of 50 µM GTP{gamma}S to the patch pipette induced irregular spikes of [Ca2+]i increase during whole-cell recordings (Fig. 6Ai; n = 8). Application of 10-s duration pulses from -75 to +5 mV significantly potentiated the GTP{gamma}S-induced Ca2+ increases (5.7-fold increase in the integral of the Ca2+ increase above the base line; n = 6, p < 0.01; Fig. 6, Aii and B). This effect of depolarization was not the result of sustained autocrine activation of P2Y1 or thromboxane A2 receptors as it was maintained during continuous perfusion with agonist-free saline and was also observed in separate experiments in the presence of either the nonspecific nucleotidase apyrase (0.32 units ml-1, n = 7) or inhibition of thromboxane A2 production by aspirin (100 µM; n = 6). Interestingly, the [Ca2+]i responses to 50 µM GTP{gamma}S (50 µM) both with (n = 9) or without (n = 5) depolarization were eliminated in P2Y1-/- megakaryocytes (Fig. 6, A, iii and iv). Although a small [Ca2+]i increase was observed at a higher concentrations of GTP{gamma}S (400 µM) in P2Y1-/- megakaryocytes, this was associated with an increased inward membrane current,3 making it difficult to further examine the effects of this activator of G-proteins. The above data do, however, show that depolarization-evoked Ca2+ increases induced by low levels of GTP{gamma}S require the presence of P2Y1 receptors.



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  		FIG. 6.
Ca2+ mobilization induced by GTP{gamma}S is voltage-dependent via a mechanism requiring P2Y1 receptors. A, intracellular application of 50 µM GTP{gamma}S induced irregular transient [Ca2+]i increases at a constant holding potential of -75 mV (i). This [Ca2+]i response was markedly potentiated by a train of 10-s duration steps to +5 mV at 0.05 Hz (ii); note that this voltage paradigm was occasionally interrupted to update capacitance and series resistance compensation. In P2Y1-/- megakaryocytes GTP{gamma}S was unable to evoke [Ca2+]i increases either alone (iii) or in combination with depolarizing pulses (iv). Traces in (i–iv) have the same timescale. B, the [Ca2+]i increase, measured as the integral (nM·s) of the response above base line, is shown for GTP{gamma}S alone (n = 8, black bar) or in combination with steps to +5mV(n = 6, white bar) (**, p < 0.01). WT, wild type.

 

Although many electrophysiological studies have used GTP{gamma}S to activate heterotrimeric G-proteins and, thus, down-stream effector pathways, [35S]GTP{gamma}S binding is commonly used to assess the proportion of activated receptors (56). This is based on the fact that the GTP analogue binds essentially irreversibly to G{alpha} subunits after receptor activation has promoted release of GDP. The effect of GTP{gamma}S on voltage-dependent Ca2+ mobilization and its absence in P2Y1-/- megakaryocytes can, therefore, be explained by a higher background activation of P2Y1 compared with other G{alpha}q-coupled receptors in the megakaryocyte. This does, however, limit the use of GTP{gamma}S as a tool for assessing an innate voltage dependence to heterotrimeric G-proteins. In contrast to GTP{gamma}S, can bind to G{alpha} subunits and activate downstream targets while GDP is still attached by mimicking the action of the {gamma} phosphate of GTP (57, 58). Perfusion of resulted in an increase in [Ca2+]i and small depolarization-evoked transients (Fig. 7A), although these were of far smaller amplitude compared with those observed in the presence of 1 µM ADP (70 ± 26 nM, n = 7, during versus 232 ± 48 nM, n = 26, during ADP; p < 0.01). This effect of was absent in P2Y1-/- megakaryocytes despite a small increase in [Ca2+]i, whereas voltage-dependent Ca2+ mobilization was observed during subsequent addition of thromboxane A2 (Fig. 7B; representative of 6 cells). Therefore, the primary site of action of membrane voltage in the control of GPCR-dependent Ca2+ mobilization is upstream of the heterotrimeric G-proteins.



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  		FIG. 7.
Stimulation of voltage-dependent Ca2+ release by aluminum tetrafluoride also requires the presence of P2Y1 receptors. Cells were exposed to the G-protein activator using a mixture of 5 mM NaF and 100 µM AlCl3. A, exposure to reversibly induced a [Ca2+]i increase and voltage-dependent Ca2+ mobilization. Subsequent application of 1 µM ADP shows the larger voltage-dependent Ca2+ responses during P2Y1 receptors. B, failed to induce voltage-dependent Ca2+ mobilization in P2Y1-/- megakaryocytes, even in cells in which depolarization potentiated Ca2+ responses induced by thromboxane A2 receptor stimulation with U46619 [GenBank] . The figures are representative of seven (A) and five (B) cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides further evidence that changes in the cell membrane potential can markedly regulate Ca2+ signaling via a number of GPCRs coupled to G{alpha}q proteins (3, 5, 7, 9, 32). The [Ca2+]i response to a step depolarization during exposure to different agonists suggests a rank order for voltage dependence of ADP > thromboxane A2 > 5-HT, which activate Ca2+ release in the platelet/megakaryocyte lineage via P2Y1, TP{alpha}, and 5-HT2A receptors, respectively (17, 29, 30, 59, 60). All are class A GPCRs (2), which contribute the largest ({approx}90%) of all known members of this superfamily. It is, therefore, likely that signaling via many other receptors within this family will show the potentially important phenomenon of voltage dependence. It was interesting that the depolarization-evoked [Ca2+]i increases in the megakaryocyte were often transient, particularly after the larger responses induced during exposure to ADP (see for example Fig. 1A). In part, this reflects the tendency for IP3-dependent Ca2+ mobilization to generate oscillatory rather than sustained [Ca2+]i increases in the megakaryocyte (14). Indeed, depolarization induces oscillatory Ca2+ responses in a proportion of cells, particularly with longer duration pulses (8). However, the transient responses to voltage could also result from an underlying ability of the voltage sensor to detect changes in membrane potential more than absolute transmembrane voltage. This may be beneficial because it would promote the detection of action potential frequency in excitable tissues. Further work is required to address this issue.

Evidence from several studies in the megakaryocyte and coronary artery smooth muscle are consistent with the hypothesis that depolarization stimulates IP3 production during activation of P2Y1 and other Gq-coupled receptors (5, 7–9, 12, 61). Thus, the voltage-dependent [Ca2+]i increase is predominantly due to IP3-dependent Ca2+ release with associated Ca2+ influx via second messenger-dependent or store-dependent pathways. It is always possible that some other as yet undetermined Ca2+-conducting pathway is also involved; however, we have previously ruled out a requirement for the one other obvious pathway, namely Na+/Ca2+ exchange (7). The fundamental role of IP3-dependent Ca2+ release explains the essential requirement for functional IP3 receptors for the response to voltage (5, 7, 12); however, this observation does not indicate the location of the voltage sensor within the GPCR cascade. The present study demonstrates that the principal voltage sensor lies upstream of phospholipase C or binding of G-protein {beta}{gamma} subunits. Furthermore, there was no evidence for an ability of voltage-gated Ca2+ channels to couple to G-protein cascades, as recently proposed in smooth and skeletal muscle (10, 11, 62, 63). It was interesting that tools commonly used to activate heterotrimeric G-proteins, and GTP{gamma}S, induced a voltage-dependent Ca2+ response and could initially be taken as evidence that the G-protein itself or its interactions with phospholipase C{beta} contribute to the response, as suggested by Ganitkevich and Isenberg (5) in their smooth muscle studies. However, the response was not observed in P2Y1 receptor-deficient megakaryocytes (see Figs. 4, 6, and 7). This can be explained by a voltage dependence to the GPCR itself together with a low background level of activation of P2Y1, but not other G{alpha}q-coupled receptors, in the megakaryocyte. GTP{gamma}S binds G{alpha} subunits as the GDP is displaced by receptor activity yet is poorly metabolized and, thus, leads to sustained activation of downstream effector responses such as IP3 generation. Thus, low background levels of P2Y1 receptor activation can account for the delay after achieving the whole-cell mode before GTP{gamma}S induces Ca2+ spiking (see Fig. 6). P2Y1 receptor activation in the absence of exogenous agonist could result from constitutive receptor activity per se, as widely described for other GPCRs (2, 64), or from autocrine activation after release of ATP and ADP, which has been described in the megakaryocyte (53, 54). In fact, we observe a very small depolarization-evoked increase in [Ca2+]i in a proportion of both rat and mouse megakaryocytes in the absence of exogenous agonist (see for example Fig. 1A, summarized in Fig. 1E), which we attribute to activation of P2Y1 receptors, as this response was completely abolished in megakaryocytes from P2Y1-/- mice.3 Unlike GTP{gamma}S, is able to activate G-proteins with GDP bound (57, 58) and, therefore, generates a [Ca2+]i increase with little delay (see Fig. 7). Basal activity within the P2Y1, but not other G{alpha}q-coupled receptors, can also account for the ability of thimerosal to induce a small voltage-dependence in wild type but not P2Y1-/- megakaryocytes. The thiol reagent is still able to elevate [Ca2+]i in P2Y1-/- megakaryocytes due to its additional action as an inhibitor of endomembrane Ca2+ATPases (52). However, to completely explain the sustained voltage dependence during GTP{gamma}S activation in wild type cells and yet complete lack of the response to depolarization during or thimerosal in the P2Y1-/- megakaryocytes, depolarization must be able to stimulate P2Y1 receptor activity in the absence of agonist. This is consistent with the emerging hypothesis that the receptor, G-protein, and effector molecules form a stable complex rather than dissociating during activation (65–68). Thus, an innate voltage dependence to the receptor can be transferred through the transduction cascade but only when the GPCR is present.

A sensitivity of the GPCR to the transmembrane potential rather than the G-protein or phospholipase C can be predicted based upon the fact that the receptor is the only protein within the transduction cascade that spans the plasma membrane. The voltage dependence could result from configurational changes in charged residues within the region of the receptor that influence G-protein activation or that control agonist binding. Additionally, voltage dependence to binding of a polar or charged agonist could contribute in an analogous manner to unblock of N-methyl-D-aspartate receptors by removal of bound Mg2+ (69). During application of exogenous agonist, voltage may act both directly on the receptor and via alterations of agonist binding; however, only the former mechanism can contribute to the potentiation of GTP{gamma}S-evoked Ca2+ release by depolarization since this was maintained after measures that reduce autocrine activation such as constant perfusion, exonucleotidases, or aspirin treatment. Furthermore, other measures that are equally effective at inducing secretion, such as phorbol ester and elevation of IP3, failed to promote voltage-dependent Ca2+ release (see Fig. 2). For the P2Y1 receptor, the regions controlling interactions with its G-proteins are undefined; however, mutagenesis has indicated a number of charged residues within the transmembrane-spanning regions that markedly influence activation by 2-MeSADP (70). These residues are postulated to be involved in agonist recognition and, thus, could be involved in a voltage dependence to ligand binding. The modulation of Ca2+ release that we observe in the absence of agonist would be expected to involve charged residues that control the efficiency with which the receptor activates G-proteins. Ben Chaim et al. (3) have recently suggested that m1 and m2 muscarinic receptors expressed in Xenopus oocytes are voltage-dependent, with depolarization exerting an opposite effect on these two receptors. From agonist binding experiments, the affinity of m2 receptors for acetylcholine is reduced, yet the affinity of m1 receptors for the agonist is enhanced by K+-induced depolarization. The authors argue that the voltage sensor resides outside the agonist binding site since this region is conserved for the two receptors and, therefore, propose that the sensor lies in the region that couples to the G-protein. We were unable to directly investigate whether membrane potential directly influences agonist binding at P2Y receptors since alterations of external K+, which would be required to set different membrane potentials in cell suspensions for radioligand binding studies, is able to modulate ADP-evoked Ca2+ mobilization under conditions where the membrane potential does not change.4

In conclusion, we have investigated the mechanism underlying the ability of membrane depolarization to potentiate Ca2+ mobilization during activation of GPCRs coupled to G{alpha}q, which could have important consequences in a variety of cell types. The evidence supports the hypothesis that the principal voltage sensor lies at the level of the receptor rather than a down-stream signaling event.


   FOOTNOTES
 
* This work was supported by British Heart Foundation Grant PG/2000108, Medical Research Council Grants G9901465 and G0301031, and by the Royal Society and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally. Back

Recipient of a Gates Cambridge Trust Scholarship and an Overseas Research Student award. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 1223-333863; Fax: 1223-333840; E-mail: mpm11{at}cam.ac.uk.

1 The abbreviations used are: GPCR, G-protein-coupled receptor; GTP{gamma}S, guanosine 5'-[{gamma}-thio]triphosphate; IP3, D-myo-inositol-1,4,5-trisphosphate; G-PIP2, 1-({alpha}-glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate; OAG, 1-oleoyl-2-acetylglycerol; U46619 [GenBank] , 9,11-dideoxy-9{alpha},11{alpha}-methanoepoxyprostaglandin F2{alpha}; DAG, diacylglycerol; P2Y1-/-, P2Y1 receptor-deficient; 5-HT, 5-hydroxytryptamine; PDGF, platelet-derived growth factor-BB; TP{alpha}, {alpha} isoform of the A2 receptor. Back

2 I. S. Gurung and M. P. Mahaut-Smith, unpublished observations. Back

3 I. S. Gurung, J. Martinez-Pinna, and M. P. Mahaut-Smith, unpublished observations. Back

4 Samantha J. Pitt, J. Martinez-Pinna, and M. P. Mahaut-Smith, unpublished observations. Back



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