Abstract
ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca(2+) through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca(2+) is universally acknowledged, the biophysics of the Ca(2+) flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca(2+) permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca(2+) flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca(2+) component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca(2+) to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca(2+) current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca(2+) through the channel.
Highlights
Ca2ϩ triggers many of the actions of extracellular ATP
When using our standard method of measuring Pf %, we saw no change in fura-2 fluorescence in mock transfected cells (n ϭ 11) in response to a concentration of agonist (6 mM) that was 10 – 60 times higher than that applied to functional recombinant P2X7 receptor (P2X7R) in our study (Fig. 2D)
Pf % was measured from HEK293 cells expressing the specific P2X7R listed in the leftmost column
Summary
Results: We measured the Ca2ϩ component of the ATP-gated non-selective cation current of P2X7 receptors. We discovered that mouse and rat splice variants with identical pore-lining sequences show significantly different Pf %, suggesting that domains that lie outside of the channel pore regulate the Ca2ϩ component of the ATP-gated current. Measurement of relative Ca2ϩ permeability typically requires a high concentration (10 –112 mM) of extracellular Ca2ϩ ([Ca2ϩ]o) that blocks the rat P2X7 current (Fig. 1A) with an IC50 of ϳ3 mM [9, 48] To circumvent this problem, we used the dye-overload method to measure the percent fractional Ca2ϩ current (Pf %) of the ATP-gated response determined in the presence of less than 2 mM free [Ca2ϩ]o [49, 50]. Reversal-based techniques do not measure the physiologically relevant Ca2ϩ current, require high concentrations of extracellular Ca2ϩ, and make the tenuous assumption that the Ca2ϩ current follows the assumptions of the Goldman-Hodgkin-Katz equation [54, 55]
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