![Figure 7. P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S1, S6) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions (n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA (n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown (n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test). (C and D) Selective pharmacological inhibition of BzATP responses by 10 µM A-438079. (C) Representative whole-cell voltage-clamp recordings show individual responses to either 300 µM ATP (top) or 300 µM BzATP (bottom) in the absence or presence of the P2X7 receptor antagonist A-438079 (preincubation >60 s; S1, S6). (D) Bar chart quantifying the efficacy of A-438079 as a function of agonist type (ATP vs. BzATP). Data are means ± SEM; normalized to control conditions. Numbers of cells are indicated above bars. Asterisk (*) denotes statistical significance, P < 0.005 (paired t test). (E) When transfected with transcript-specific siRNA (green), posttranscriptional gene silencing of P2X7 expression is confirmed by quantitative PCR. Relative transcript levels (means ± SEM) are normalized to mRNA quantities in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Asterisks (*) denote statistical significance, P < 0.005 (one-way ANOVA with Tukey’s HSD post hoc test). (F) Representative current density plot over time. Each dot represents current measured at −80 mV obtained from sequential voltage ramps as in G and H. Data correspond to spermatogonia transfected with either targeting P2X7 siRNA (green) or a nontargeting control construct (black). When challenged with 1 mM ATP, a fast but relatively small inward current develops and shows a transient peak (first Imax; red arrowhead). After apparent desensitization, current amplitudes gradually increase over tens of seconds until a secondary maximum is reached (second Imax; red asterisk). (G and H) Exemplary current–voltage curves (−100 to 100 mV; 250 ms; 1 Hz repetition; S1, S6) in response to 1 mM ATP, corresponding to time points (first G vs. second H Imax) as indicated in F (red arrowhead and asterisk). Color code as for E and F. Data were corrected by digital offline subtraction of averaged leak controls recorded before stimulation. (I and J) Quantification of mean current densities measured at −80 mV at first (I) versus second (J) Imax; see F–H. Bar graphs display means ± SEM. Numbers of cells as shown above bars. Asterisks (*) indicate statistical significance, P < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test).](https://cdn.rupress.org/rup/content_public/journal/jgp/148/3/10.1085_jgp.201611636/5/jgp_201611636_fig7.jpeg?Expires=1773552390&Signature=gFY0uq-sWJPfhxOhTEwjhHOzQOq6t5NBuAivWm8X0THpcfIgtL2VZgel4zfUf9hsV8QlehpqW5Y682v3hhakJrfrB7CT5HI4N6Edtm0cd04IkszXf6quP9XehUziNJzrCCdwe5qldHBnkclMaEPsd-ZR-lgGjpvZXXYGyvukXx7HjO5DlozXzaoajilyk2dJAjJVLnp2FL434JF3sQB05JRzT1WTiyPQ2D4HN3TqqSmyjsuQevQvgtqm1s84NzaJucV0Jer7l58DJGNnIr61YGp4N4cuTmhR436mqaNoclaUoxL1wpOKuO1LTRaH2uGaWKJ9GC7K9uDD8vXsRvVd3Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S1, S6) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions (n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA (n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown (n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test). (C and D) Selective pharmacological inhibition of BzATP responses by 10 µM A-438079. (C) Representative whole-cell voltage-clamp recordings show individual responses to either 300 µM ATP (top) or 300 µM BzATP (bottom) in the absence or presence of the P2X7 receptor antagonist A-438079 (preincubation >60 s; S1, S6). (D) Bar chart quantifying the efficacy of A-438079 as a function of agonist type (ATP vs. BzATP). Data are means ± SEM; normalized to control conditions. Numbers of cells are indicated above bars. Asterisk (*) denotes statistical significance, P < 0.005 (paired t test). (E) When transfected with transcript-specific siRNA (green), posttranscriptional gene silencing of P2X7 expression is confirmed by quantitative PCR. Relative transcript levels (means ± SEM) are normalized to mRNA quantities in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Asterisks (*) denote statistical significance, P < 0.005 (one-way ANOVA with Tukey’s HSD post hoc test). (F) Representative current density plot over time. Each dot represents current measured at −80 mV obtained from sequential voltage ramps as in G and H. Data correspond to spermatogonia transfected with either targeting P2X7 siRNA (green) or a nontargeting control construct (black). When challenged with 1 mM ATP, a fast but relatively small inward current develops and shows a transient peak (first Imax; red arrowhead). After apparent desensitization, current amplitudes gradually increase over tens of seconds until a secondary maximum is reached (second Imax; red asterisk). (G and H) Exemplary current–voltage curves (−100 to 100 mV; 250 ms; 1 Hz repetition; S1, S6) in response to 1 mM ATP, corresponding to time points (first G vs. second H Imax) as indicated in F (red arrowhead and asterisk). Color code as for E and F. Data were corrected by digital offline subtraction of averaged leak controls recorded before stimulation. (I and J) Quantification of mean current densities measured at −80 mV at first (I) versus second (J) Imax; see F–H. Bar graphs display means ± SEM. Numbers of cells as shown above bars. Asterisks (*) indicate statistical significance, P < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test).
