Figure 1.
Purified histone octamer proteins activate P2XR7 currents. (A) Representative traces of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. Cells were held at −60 mV. 1 s voltage ramps from −60 to 40 mV were performed at baseline and in the presence of 600 μM ATP or 10 μg/ml histone protein in high Ca2+ Ringer’s solution. (B and C) Average whole-cell current at −60 mV in oocytes 24–48 h after nuclear injection of mouse (B, “m.”) or human (C, “h.”) P2XR7 cDNA in response to 600 μM ATP, 50 μM BzATP, or 10 μg/ml histones (uninjected cells, “Ctrl”; Ave ± SEM, n = 3–9 animals, 8–28 cells). (D) ATP-, BzATP-, and histone-evoked m.P2XR7 current decay with agonist removal estimated by fitting data from A (highlighted in gray) to a single exponential (Ave ± SEM, n = 3 animals, 3–10 cells, * P = 0.0016 histone vs. BzATP, * P < 0.0001 histone vs. ATP). (E and F) Representative current–voltage relationships measured once peak sustained currents were achieved in A (n = 3 animals, 10 cells). (G and H) Average reversal potential (G) and whole-cell conductance (H) for ATP- and histone-evoked currents in oocytes expressing P2XR7 calculated from voltage ramps in E and F (Ave ± SEM, n = 3 animals, 10 cells). (I and J) average ATP- or histone-evoked inward current measured at different holding potentials (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −24 mV for ATP and −39 mV for histone). Gray line represents best fit linear regression. (J) Histone-evoked currents were normalized to ATP-evoked currents from the same cell (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −35 mV). (K) Average whole-cell current in P2XR7-expressing cells in response to histone concentrations from 1 to 10 μg/ml and in response to boiled and Pro K digested histone protein at 10 μg/ml (Pro K–treated histones also boiled to inactivate Pro K, Ave ± SEM, n = 3–9 animals, 8–20 cells, * P < 0.0001 by non-parametric one-way ANOVA).

Purified histone octamer proteins activate P2XR7 currents. (A) Representative traces of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. Cells were held at −60 mV. 1 s voltage ramps from −60 to 40 mV were performed at baseline and in the presence of 600 μM ATP or 10 μg/ml histone protein in high Ca2+ Ringer’s solution. (B and C) Average whole-cell current at −60 mV in oocytes 24–48 h after nuclear injection of mouse (B, “m.”) or human (C, “h.”) P2XR7 cDNA in response to 600 μM ATP, 50 μM BzATP, or 10 μg/ml histones (uninjected cells, “Ctrl”; Ave ± SEM, n = 3–9 animals, 8–28 cells). (D) ATP-, BzATP-, and histone-evoked m.P2XR7 current decay with agonist removal estimated by fitting data from A (highlighted in gray) to a single exponential (Ave ± SEM, n = 3 animals, 3–10 cells, * P = 0.0016 histone vs. BzATP, * P < 0.0001 histone vs. ATP). (E and F) Representative current–voltage relationships measured once peak sustained currents were achieved in A (n = 3 animals, 10 cells). (G and H) Average reversal potential (G) and whole-cell conductance (H) for ATP- and histone-evoked currents in oocytes expressing P2XR7 calculated from voltage ramps in E and F (Ave ± SEM, n = 3 animals, 10 cells). (I and J) average ATP- or histone-evoked inward current measured at different holding potentials (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −24 mV for ATP and −39 mV for histone). Gray line represents best fit linear regression. (J) Histone-evoked currents were normalized to ATP-evoked currents from the same cell (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −35 mV). (K) Average whole-cell current in P2XR7-expressing cells in response to histone concentrations from 1 to 10 μg/ml and in response to boiled and Pro K digested histone protein at 10 μg/ml (Pro K–treated histones also boiled to inactivate Pro K, Ave ± SEM, n = 3–9 animals, 8–20 cells, * P < 0.0001 by non-parametric one-way ANOVA).

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