Functional and physical interaction between P2Y receptors and neuronal ion channels

Neuronale P2Y-Rezeptoren gehören zur Familie der nukleotidsensitiven G-Protein-gekoppelten Rezeptoren (GPCRs) und sind bekannt dafür, dass sie spannungsgesteuerte Ionenkanäle, insbesondere Kv7-Kalium- und Cav2.2-Calciumkanäle, modulieren. In vielen Fällen hängt diese Modulation vom Vorhandensein bzw. Fehlen von so genannten Gerüstproteinen ab, die Rezeptoren und Ionenkanäle in unmittelbare Nähe bringen. Diese Nähe erleichtert wiederum die Interaktion und garantiert eine effiziente G-Protein-vermittelte Modulation. Dieses Projekt untersucht, ob eine enge Kolokalisierung zwischen P2Y-Rezeptoren und Ionenkanälen eine Voraussetzung für deren funktionelle Interaktion ist. Jeder der untersuchten P2Y-Rezeptoren und Ionenkanälen wurden fluoreszierend markiert und transient in tsA201 Zellen ko-transfiziert. Elektrophysiologische Messungen ermöglichten die Bestimmung der Kanalmodulation durch Nukleotide. Die Aktivierung von P2Y1, aber nicht von P2Y12-Rezeptoren durch ADP hemmte K+ Ströme in konzentrationsabhängiger Weise um bis zu 20,5 1,9%. Umgekehrt, verringerte die Aktivierung beider Rezeptoren, P2Y1 und P2Y12, Ca2+ Ströme jeweils um bis zu 60.1 7.4% und 76.3 4.2%. Zur Bestimmung der Protein-Protein-Interaktion zwischen Rezeptoren und Kanälen wurden FRET (Förster resonance energy transfer) und DRAP (donor recovery after acceptor photobleaching) verwendet. Diese Experimente zeigen, dass P2Y1 eine Protein-Protein-Interaktion sowohl mit Kv7.2/7.3 (NFRET 0.32 0.02, DRAP 10.3 3.0%) als auch mit Cav2.2 (NFRET 0.37 0.02, DRAP 10.1 1.8%) aufweist. Andererseits, interagiert P2Y12 nur mit Cav2.2 (NFRET 0.39 0.03, DRAP 12.7 1.0%), jedoch nicht mit Kv7.2/7.3-Kanälen. Um die Mobilität der Rezeptoren und Kanäle innerhalb der Membran zu untersuchen, wurde FRAP (fluorescence recovery after photobleaching) angewendet. Diese Experimente zeigten, dass Ionenkanäle alleine schneller sind als Rezeptoren. Die Koexpression von P2Y-Rezeptoren verlangsamte die Mobilität von Cav2.2 signifikant um 50% (von 3,3 s auf 6,2 s). Im Fall von Kv7.2/7.3 wurden die Werte durch die Anwesenheit von P2Y Rezeptoren nicht signifikant verändert. Somit folgt, dass die funktionelle Modulation von Kv7 durch P2Y1, und von Cav2.2 durch P2Y1- und P2Y12 Rezeptoren auf eine unmittelbare Nähe von Rezeptoren und Kanälen beruht. Im Fall von Cav2.2 und P2Y12 wird dies noch durch eine physische Interaktion zusätzlich bekräftigt.Neuronal P2Y receptors belong to the family of nucleotide-sensitive G-protein coupled receptors (GPCRs) and are known to modulate voltage-gated ion channels, especially Kv7 potassium and Cav2.2 calcium channels. It has been reported that in many cases, this modulation relies on the presence or absence of so-called scaffold proteins which bring GPCRs and ion channels in close proximity. This tight juxtaposition of the proteins is believed to facilitate the interaction and to guarantee efficient G-protein-mediated modulation. This project evaluates whether a tight colocalization between P2Y receptors and ion channels is a requirement for their functional interaction. Therefore, fluorescently labelled P2Y receptors and ion channels were co-transfected transiently into tsA201 cells for electrophysiological and microscopic studies. Electrophysiological measurements showed that the activation of P2Y1, but not of P2Y12 receptors by ADP inhibited the K+ currents in a concentration-dependent manner by up to 20.5 1.9%. Contrarily, activation of both P2Y1 and P2Y12 receptors reduced the Ca2+ currents by up to 60.1 7.4% and 76.3 4.2%, respectively. The protein-protein interactions between receptors and channels were determined by Förster resonance energy transfer (FRET) and donor recovery after acceptor photobleaching (DRAP). Results show that P2Y1 has a protein-protein interaction with both Kv7.2/7.3 (NFRET 0.32 0.02, DRAP 10.3 3.0%) and Cav2.2 (NFRET 0.37 0.02, DRAP 10.1 1.8%) channels. On the other hand, P2Y12 has an interaction only with Cav2.2 (NFRET 0.39 0.03, DRAP 12.7 1.0%) but not with the Kv7.2/7.3 channels. The mobility of the receptors and channels within the membrane was elucidated by fluorescence recovery after photobleaching (FRAP). Here, we found that the mobility of the ion channels alone is higher than that of the receptors. Coexpression of the P2Y receptors significantly slowed down the mobility of the Cav2.2 channel by 50% (from 3.3 s to 6.2 s). In the case of Kv7.2/7.3 channels, the values were not significantly changed by the presence of P2Y. Thus, we conclude that the functional control of Kv7 by P2Y1 and Cav2.2 by P2Y1 and P2Y12 receptors relies on their close apposition within the membrane, while in the case of Cav2.2 and P2Y12 this is even accompanied by a physical interaction.submitted by Hend GafarZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeB

Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP

ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.W 1205-B09(VLID)309167