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Figure 1. Structure and function of four homotetrameric configurations of high-affinity Ca2+ sensors used to investigate cooperativity among Ca2+ sensors. (A) Cartoon of a single WT subunit (upper left) and a schematic diagram of a WT channel formed from four of these subunits (upper right). Each WT subunit has two high-affinity Ca2+ sensors on the intracellular COOH terminus, an RCK1 sensor (R) and a Ca-bowl sensor (B), giving an (RB) subunit. WT channels are comprised from four such subunits, designated 4(RB). WT channels were fully activated with 100 μM Ca2+i at + 50 mV, as indicated by the single-channel current record (upper current trace). Addition of 1.5 mM extracellular TEA, (TEAO) completely blocked the currents (lower trace). Arrows indicate the closed current level. (B) Mutation of the RCK1 site produces a ÎB subunit, where Î indicates a mutated sensor. Homomeric 4(ÎB) channels expressed from ÎB subunits have greatly reduced activation with 100 μM Ca2+i (upper current trace) and can be blocked by 1.5 mM TEAO. (C) Mutation of the Ca-bowl site produces an RÎ subunit. Homomeric 4(RÎ) channels expressed from RÎ subunits also have greatly reduced activation with 100 μM Ca2+i. The channel is not blocked by TEAO because of the presence of a Y294V pore mutation (lower trace). (D) Mutation of both the RCK1 sensor and the Ca bowl produce ÎÎ subunits. Homomeric 4(ÎÎ) channels are Ca2+ insensitive up to 1,000 μM Ca2+. The channel is not blocked by TEAO because of the presence of a Y294V pore mutation (lower trace). Current traces in this and the following figures are representative excerpts from longer records.
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Figure 2. RCK1 sensors and Ca bowls are approximately equivalent in activating BK channels when distributed on separate subunits. Po vs. Ca2+i plots for 4(RB), 4(RÎ), 4(ÎB), 2(ÎB)+2(RÎ), and 4(ÎÎ) channels. Data for each plotted point is from 5â7 single-channel patches. The lines are fits with the Hill equation (Eq. 1). The data from the 4(RÎ), 4(ÎB), and 2(ÎB)+2(RÎ) channels cluster together, and consequently these data were simultaneously fit to obtain the continuous line through the data. The Kd for the 4(RB) and combined 4(RÎ), 4(ÎB), and 2(ÎB)+2(RÎ) channels are 2.01 and 94.4 μM, respectively, with Hill coefficients of 3.30 and 1.05, respectively. In this and the following figures data that were obtained for a free Ca2+ ⤠0.01 μM are plotted at 0.01 μM to save space, as there was no difference in response at such low Ca2+. Points at 0.01 have been displaced slightly so they can be seen.
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Figure 3. Identification of the subunit stoichiometry of the heteromeric channels used to explore intrasubunit cooperativity. (A) Presentation of the channels examined in this study, their channel designation, and distributions of the high-affinity Ca2+ sensors on the subunits, where blue squares are RCK1 sensors and red hexagons are Ca bowls. For 2(RB)+2(ÎÎ) and 2(ÎB)+2(RÎ) channels, both adjacent and diagonal subunit configurations are shown. (B) 2(RB)+2(ÎÎ) channels were obtained by coexpressing RB subunits together with ÎÎ subunits that had the Y294V pore mutation to relieve TEAO block. Each expressed channel had one of five distinct current amplitudes in the presence of 1.5 mM TEAO. Examples of single-channel currents (+50 mV) from channels with one of the four nonzero current amplitudes are presented. (An example of the zero current amplitude is presented in Fig. 1 A where the channel was first identified in the absence of TEAO.) The deduced subunit stoichiometry for each of the current amplitudes is shown at the right, with both possible configurations for the 2(RB)+2(ÎÎ) channel shown. The 2(RB)+2(ÎÎ) channel can be identified by a current level of â¼5 pA at + 50 mV. (C) Plot of single-channel current amplitude against the number of ÎÎ subunits, assuming that the current amplitude is proportional to the number of subunits with the pore site mutation that removes TEAO block. (D and E) 2(ÎB)+2(RÎ) channels were obtained by coexpressing (ÎB) subunits together with RÎ subunits that had the Y294V pore mutation that relieved TEAO block. 2(ÎB)+2(RÎ) channels were identified by a current level of â¼5 pA at + 50 mV, following the same strategy as used above.
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Figure 4. Two high-affinity Ca2+ sensors on the same subunit are more effective than when on different subunits, indicating intrasubunit cooperativity. (A and B) Representative single-channel currents recorded from a 2(RB)+2(ÎÎ) channel (A) and a 2(ÎB)+2(RÎ) channel (B) at four different Ca2+i at +50 mV. The Po is higher at each level of Ca2+i when two of the four subunits have two high-affinity Ca2 sensors each, rather than when each of the four subunits has a single high-affinity Ca2+ sensor. TEAO (1.5 mM) was present for both channels. (C) Plots of Po vs. Ca2+i for the indicated channel types: 2(RB)+2(ÎÎ) channels (filled diamonds, Kd = 29.7μM, Hill coefficient = 1.35) require less Ca2+i for the same Po than 2(ÎB)+2(RÎ) channels (open circles, Kd = 114.4 μM, Hill coefficient = 1.04). The dashed line is the response for WT channels from Fig. 2. Data for each plotted point is from 5â7 single-channel patches.
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Figure 5. A gating model incorporating both intra- and intersubunit cooperativity (Scheme 2) can describe the Ca2+-dependent activation of BK channels comprised of subunits with different numbers and distributions of high-affinity Ca2+ sensors. (A) Po vs. Ca2+i plots for the six different channel types, as indicated. The continuous lines are simultaneous fits with Scheme 2 to all the plotted data and were calculated with L(V) = 2500 (from Niu and Magleby, 2002), KBC = 6.3 μM, KBO = 1.0 μM, KRC = 7.8 μM, KRO = 1.3 μM, KMC = 3000 μM; KMO = 644 μM, W = 1.19, Pmax = 0.90. An assumption that the two high-affinity Ca2+ sites were identical gave an equally good fit (not shown) with L(V) = 2500, KBC = KRC = 6.9 μM, KBO = KRO = 1.1 μM, KMC = 3000 μM, KMO = 644 μM, W = 1.19. The values of n for each curve were set by the subunit composition (see text). KMC was poorly defined and was set to the same value of both fits. (B) Po vs. Ca2+i plots for the data from Niu and Magleby (2002) for BK channels with different numbers of Ca bowls. From left to right the subunit composition was 4(RB), 3(RB)+1(RÎ), 2(RB)+2(RÎ), 1(RB)+3(RÎ), and 4(RÎ). The continuous lines are simultaneous fits with Scheme 2 to all the data in part B, and were calculated with L(V) = 2500, KBC = 7.18 μM, KBO = 1.00 μM, KRC = 110.35 μM, KRO = 18.98 μM, KMC = 3000 μM; KMO = 961 μM, W = 1.63, and Pmax = 0.95.
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Figure 6. If the assumptions are made that the high-affinity Ca2+ sensors act at a flexible interface and that the interfaces between RCK domains in the gating ring alternate between fixed and flexible, then the data are consistent with a flexible, rather than a fixed, interface between the RCK1 and RCK2 domains of a single subunit. Schematic diagrams of WT BK channels and their gating rings are presented with different distributions of the high-affinity Ca2+ sensors assuming (A1âA3) a flexible intrasubunit interface or (B1âB3) a fixed intrasubunit interface. Transmembrane segments S0âS5 are removed in the side views of the BK channels in A1 and B1 to show the S6 gates. RCK1 domains are ellipses, RCK2 domains are squares, RCK1 sensors are blue squares, Ca bowls are red hexagons, RCK domains from the same subunit are of the same color, fixed interfaces are indicated by a bar across the interface, and flexible interfaces are indicated by contact between the RCK1 and RCK2 domains. The distributions of the RCK1 sensors and Ca bowls on the subunits are indicated in the stick diagrams for each channel type. The four channel types in A2 (for flexible intrasubunit interfaces) had similar Ca2+ response curves and required 3.9-fold more Ca2+ for half activation than the channel types in A3. These same channel types are repeated in B2 and B3 for fixed intrasubunit interfaces. The consistent distributions of high-affinity Ca2+ sensors in A2 and A3 assuming a flexible intrasubunit interface can be contrasted to the inconsistent distributions in B2 and B3 assuming a fixed intrasubunit interface, suggesting a flexible intrasubunit interface.
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