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

Juan Martinez-Pinna,Iman S Gurung,Catherine Vial,Catherine Leon,Christian Gachet,Richard J Evans,Martyn P Mahaut-Smith

doi:10.1074/jbc.m407783200

Juan Martinez-Pinna, Iman S Gurung + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m407783200

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Abstract

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 Galphaq-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 Cgamma-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 betagamma 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 Galphaq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPgammaS dialysis and to a lesser extent during AlF4- or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Galphaq-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.

Highlights

Emerging evidence suggests that Ca2؉ release evoked by certain G-protein-coupled receptors can be voltage-dependent; 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␣q-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP
Voltage-dependent Ca2ϩ Release during Activation of Multiple G␣q-coupled G-protein-coupled receptors (GPCRs)—The megakaryocyte is a useful cell type for studying voltage control of GPCR-evoked Ca2ϩ signaling as this electrically inexcitable cell type expresses multiple surface receptors known to couple to Ca2ϩ mobilization in the

Summary

EXPERIMENTAL PROCEDURES

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 mlϪ1 type V or type VII apyrase. For assessment of the relative voltage dependence to different receptors, we selected agonist concentrations that generated a robust Ca2ϩ 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.. 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 [Ca2ϩ]i transients This approach may have introduced some variation in internal Ca2ϩ store content as a consequence of different driving forces for Ca2ϩ influx, it avoided the voltage-dependent Ca2ϩ responses caused by changing the holding potential during activation of the G-protein-coupled receptor. 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

Although many electrophysiological studies have used

DISCUSSION