P2X7 reseptörlerinin aktive ettiği porların geçirgenlik özelliklerinin incelenmesi

P2X7 bir katyon kanalidir ve buyuk molekullere gecirgen olan secici olmayan buyuk bir por olusturdugu bilinmektedir. Bu calismada, HEK-K4 (kalici olarak sican P2X7 reseptoru eksprese ettirilen) ve RAW 264.7 (endojen olarak fare P2X7 reseptoru tasiyan) hucrelerinde farkli yuk ve molekul agirliktaki floresan boyalar kullanarak, P2X7 reseptorunun aktive ettigi porun gecirgenlik ozellikleri ve hucre ici kalsiyumun bu porun aktivasyonundaki rolunu inceledik. Bu boyalar pozitif yuklu Ethidium Bromide, YO-PRO-1, YO-YO-1, TO-TO-1 ve negatif yuklu Lucifer Yellow, Calcein.Hucre disi kalsiyum (1 mM) varliginda, P2X7 aktivasyonundan sonra HEK-K4 hucreleri YO-PRO-1 ve Lucifer Yellow boyalarini gecirdi. Ilginc olarak, hucredisi kalsiyum yokken Lucifer Yellow girisi ortadan kalkarken YO-PRO-1 girisi etkilenmedi. YO-PRO-1 girisi hucreici kalsiyumun celasyonuyla da bloke olmadi. RAW 264.7 hucrelerinde, hucre disinda kalsiyum yokken ATP uygulanmasi YO-PRO-1 ve Lucifer Yellow girisini indukledi. RAW hucrelerinde, hucreici kalsiyumun celasyonuyla bu boyalarin girisinin engellenmedigini de bulduk. ATP uygulamasi olmaksizin hucreici kalsiyum bir iyonoforla ((Br-A23187 10 uM) dogrudan arttirildiginda, HEK-K4 hucrelerinde Lucifer Yellow, RAW 264.7 hucrelerinde de YO-PRO-1 ve Lucifer Yellow girisi gozledik. Bu bulgular HEK-K4 ve RAW 264.7 hucrelerinde P2X7 reseptorunun aktive ettigi buyuk floresan boyalarin girisinden sorumlu farkli secicilik ozelligi olan birden fazla yolagin oldugunu gostermistir. Bu yolaklar hucreici kalsiyumuna olan bagimliliklariyla ayrilabilir.AbstractP2X7 is a cation channel and has been known to form a non-selective large ?pore? which is permeable to large molecules. In this study, we investigated permeability properties of P2X7-activated pores and role of intracellular [Ca2+] in activation of these pore in HEK-K4 (stabily expresses rat P2X7 receptor) and RAW 264.7 (endogenly expresses mouse P2X7 receptor) cells by using fluorescence tracers with different charges and molecular weights. These dyes are positively charged Ethidium Bromide, YO-PRO-1, YO-YO-1, TO-TO-1 and negatively charged Lucifer Yellow, Calcein. In the presence of extracellular Ca2+ (1mM), after P2X7 stimulation HEK-K4 cells permeates YO-PRO-1 and also Lucifer Yellow dyes. Interestingly, in the absence of extracellular Ca2+ Lucifer Yellow uptake disappeared but YO-PRO-1 did not and also YO-PRO-1 uptake was not blocked by intracellular Ca2+ chelation. In RAW 264.7 cells ATP application induced both YO-PRO-1 and Lucifer Yellow uptake in the absence of extracelluler Ca2+. We also found that in RAW cells uptake of these dyes were not supressed by intracellular Ca2+ chelation. When intracellular [Ca2+] is increased directly by an ionophore (Br-A23187 10 uM), without ATP application, uptake of Lucifer Yellow in HEK-K4 and uptake of YO-PRO-1 and Lucifer Yellow in RAW cells is activated. These findings show that more than one pathway with different permeant selectivity is responsible for the P2X7 activated uptake of large fluorescent tracers in RAW 264.7 and HEK-K4 cells. Also these pathways can be seperated by their dependency on intracellular Ca2+.