P2X7 receptor activates multiple selective dye-permeation pathways in RAW 264.7 and human embryonic kidney 293 cells
Serife Cankurtaran-Sayar1, Kemal Sayar, Mehmet Ugur
1Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey.
Abstract:
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.
Insights
The P2X7 receptor activates at least two distinct cellular pathways for dye uptake, challenging the idea of a single nonselective pore. These pathways show different properties for cationic and anionic dyes.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Immunology
Background:
- The P2X7 receptor is a significant drug target due to its role in cellular responses.
- P2X7 receptor stimulation triggers the uptake of large molecules into cells.
- The exact mechanism of this P2X7 receptor-activated permeability pathway remains unclear, with a single nonselective pore often hypothesized.
Purpose of the Study:
- To investigate the mechanisms of P2X7 receptor-mediated dye uptake in human embryonic kidney (HEK) 293 cells and RAW 264.7 macrophages.
- To determine if P2X7 receptor activation leads to a single nonselective pore or multiple distinct pathways.
- To characterize the properties and ion dependency of these P2X7 receptor-activated permeation pathways.
Main Methods:
- Utilized confocal microscopy to visualize and quantify the uptake of various fluorescent dyes (YO-PRO-1, TO-TO-1, lucifer yellow, calcein, ethidium) into HEK-rP2X7 and RAW 264.7 cells.
- Stimulated P2X7 receptors using ATP application.
- Assessed the role of intracellular calcium concentration ([Ca2+](i)) in pathway activation.
Main Results:
- P2X7 receptor stimulation in HEK-rP2X7 cells activated two pathways: one for cationic dyes (YO-PRO-1, TO-TO-1) independent of [Ca2+](i), and another for anionic dye lucifer yellow dependent on [Ca2+](i).
- In RAW 264.7 cells, P2X7 receptor stimulation also induced two pathways: one for ethidium and another for lucifer yellow, with no clear [Ca2+](i) dependence observed.
- These findings indicate distinct permeation properties for cationic and anionic dyes.
Conclusions:
- P2X7 receptor activation does not form a single nonselective pore.
- At least two distinct permeation pathways are activated by the P2X7 receptor: one for cationic dyes and one for anionic dyes.
- These pathways exhibit different activation characteristics, particularly concerning intracellular calcium levels.
