![Figure 2. The effects of luminal Ca2+ on the response of the RYR2 channel to ATP. (A) Representative current traces of single RYR2 activity at various cytosolic ATP concentrations in the presence of 0.005 (top), 8 (middle), and 53 mM [Ca2+]L (bottom). In all three datasets, [Ca2+]C was 100 nM. Channel openings are in the upward direction. Dashes at the left of the traces indicate the closed state of the channel. Please note the different current calibration and different range of ATP concentrations. (B) The ATP dependence of RYR2 PO (top) in the presence of 0.005, 1, 8, 15, 26, and 53 mM [Ca2+]L (black squares; open circles; black, gray, and open triangles; and black diamonds, respectively). Solid lines are Hill equation fits. The whole dataset at 1–53 mM Ca2+ was fitted simultaneously when nH and EC50 as fitted parameters were shared while POmax was free and POmin was set to the average PO in the absence of ATP. The data in the virtual absence of luminal Ca2+ were fitted separately. The luminal Ca2+ dependence of PO (middle) in the absence of ATP (POmin, open circles) and of the maximum achievable PO in the presence of ATP (POmax, black circles). Solid line is the best fit of the relationship between POmax and [Ca2+]L by the Hill equation, with apparent affinity for luminal Ca2+ of 14.3 ± 2.0 mM. (Bottom) The luminal Ca2+ dependence of EC50 for ATP. Data are presented as average ± SEM. Asterisks denote a significant increase of POmin (P < 0.05). (C and D) The average open (tO) and closed times (tC) determined on 30-s intervals for 0.005, 1, 8, 15, 26, and 53 mM [Ca2+]L (black squares; open circles; black, gray, and open triangles; and black diamonds, respectively) are displayed as a function of either PO (C) or ATP concentration (D). Insets in C show the rising phase of tO versus PO and tC versus PO plots on an expanded scale (PO ranged from 0 to 0.02). In C and D, labels next to the points specify the conditions of the experiments (L0.005, L1, L8–26, and L53 for [Ca2+]L of 0.005, 1, 8–26, and 53 mM, respectively). Error bars in C and D are shown only when the SEM is larger than symbol size.](https://cdn.rupress.org/rup/content_public/journal/jgp/140/2/10.1085_jgp.201110708/4/jgp_201110708_fig2.jpeg?Expires=1767688275&Signature=vd2Aj8~iFDIWXyylzdKhZRnBYUCOZXzgvdVjHCS0sIhQEd3xUA4HIbGn4aLNqiJpMGudRk8ihO8ZvWoNwYQNqiNHpBEg2urCQG~vPXnY4sg60Ar4LAlI7jqgJ8Oqrx3fQzXHTNhfndMah-6TQzmOJZDn89aLxJSWKPZDgfmGj5ZGvBRGRz5E6Y9fvmPFE1dBWM11kSkTs3xcw0Yz2Jq3zVjDh1hdnLIXKSH8pl1iZ9WhlnskTy5K-ncgCCwXmk0Q6Cmg3G5vz99jLKZ6PQxCgXvb6308DuYFKO7oYL4h2TuWqyZ5X9Zb3YLKCVnZRG0j1PMieU8UFuLC37S9n-4NPw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The effects of luminal Ca2+ on the response of the RYR2 channel to ATP. (A) Representative current traces of single RYR2 activity at various cytosolic ATP concentrations in the presence of 0.005 (top), 8 (middle), and 53 mM [Ca2+]L (bottom). In all three datasets, [Ca2+]C was 100 nM. Channel openings are in the upward direction. Dashes at the left of the traces indicate the closed state of the channel. Please note the different current calibration and different range of ATP concentrations. (B) The ATP dependence of RYR2 PO (top) in the presence of 0.005, 1, 8, 15, 26, and 53 mM [Ca2+]L (black squares; open circles; black, gray, and open triangles; and black diamonds, respectively). Solid lines are Hill equation fits. The whole dataset at 1–53 mM Ca2+ was fitted simultaneously when nH and EC50 as fitted parameters were shared while was free and was set to the average PO in the absence of ATP. The data in the virtual absence of luminal Ca2+ were fitted separately. The luminal Ca2+ dependence of PO (middle) in the absence of ATP (, open circles) and of the maximum achievable PO in the presence of ATP (, black circles). Solid line is the best fit of the relationship between and [Ca2+]L by the Hill equation, with apparent affinity for luminal Ca2+ of 14.3 ± 2.0 mM. (Bottom) The luminal Ca2+ dependence of EC50 for ATP. Data are presented as average ± SEM. Asterisks denote a significant increase of (P < 0.05). (C and D) The average open (tO) and closed times (tC) determined on 30-s intervals for 0.005, 1, 8, 15, 26, and 53 mM [Ca2+]L (black squares; open circles; black, gray, and open triangles; and black diamonds, respectively) are displayed as a function of either PO (C) or ATP concentration (D). Insets in C show the rising phase of tO versus PO and tC versus PO plots on an expanded scale (PO ranged from 0 to 0.02). In C and D, labels next to the points specify the conditions of the experiments (L0.005, L1, L8–26, and L53 for [Ca2+]L of 0.005, 1, 8–26, and 53 mM, respectively). Error bars in C and D are shown only when the SEM is larger than symbol size.
