P2X7 receptor activates multiple selective dye-permeation pathways in RAW 264.7 and human embryonic kidney 293 cells.
P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this P2X7 receptor-activated permeability pathway. We examined human embryonic kidney (HEK) 293 cells transfected with rat P2X7 receptors (HEK-rP2X7) and a macrophage derived cell line, RAW 264.7, that expresses an endogenous P2X7 receptor. We used confocal microscopy to investigate uptake of different types of dyes into these cells after ATP application. Stimulation of P2X7 receptors in HEK-rP2X7 cells activated two different dye uptake pathways. The first was permeable to the cationic fluorescent dyes YO-PRO-1 and TO-TO-1 but not to the anionic dyes lucifer yellow and calcein and did not require intracellular Ca2+ concentration ([Ca2+](i)) increase to be activated. The second pathway permeated only lucifer yellow and was completely dependent on [Ca2+](i) for activation. In RAW 264.7 cells, P2X7 receptor stimulation activated uptake of ethidium, YO-PRO-1, TO-TO-1, lucifer yellow, and calcein. Again, two different permeation pathways were discerned in RAW 264.7 cells: one permeated only ethidium and the other one, only lucifer yellow. We did observed no clear [Ca2+](i) dependence for these permeation pathways. Our results demonstrate that instead of a single nonselective pore, P2X7 receptor seems to activate at least two permeation pathways, one for cationic and one for anionic dyes with different activation properties.
Cankurtaran-Sayar
Serife
S
Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey.
Sayar
Kemal
K
Ugur
Mehmet
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001583:Benzoxazoles / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000187:drug effects* / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D015533:Transcriptional Activation; D014162:Transfection
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D007546:Isoquinolines; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; D000255:Adenosine Triphosphate; C017475:lucifer yellow; D002118:Calcium