Intra- and intersubunit cooperativity in activation of BK channels by Ca2+

Abstract

The activation of BK channels by Ca(2+) is highly cooperative, with small changes in intracellular Ca(2+) concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca(2+). Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca(2+) sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K(+)) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca(2+) sensors, we examine channels with eight different configurations of functional high-affinity Ca(2+) sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca(2+) activation of the channel when there is only one high-affinity Ca(2+) sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca(2+) sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca(2+) sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca(2+) sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca(2+) response for eight possible subunit configurations of the high-affinity Ca(2+) sensors as well as for three additional configurations from a previous study.

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, (TEA_O) completely blocked the currents (lower trace). Arrows indicate the closed current level. (B) Mutation of the RCK1 site produces a ΔB subunit, where Δ indicates a mutated sensor. Homomeric 4(ΔB) channels expressed from ΔB subunits have greatly reduced activation with 100 μM Ca²⁺ _i (upper current trace) and can be blocked by 1.5 mM TEA_O. (C) Mutation of the Ca-bowl site produces an RΔ subunit. Homomeric 4(RΔ) channels expressed from RΔ subunits also have greatly reduced activation with 100 μM Ca²⁺ _i. The channel is not blocked by TEA_O because of the presence of a Y294V pore mutation (lower trace). (D) Mutation of both the RCK1 sensor and the Ca bowl produce ΔΔ subunits. Homomeric 4(ΔΔ) channels are Ca²⁺ insensitive up to 1,000 μM Ca²⁺. The channel is not blocked by TEA_O 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.

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(RΔ), 4(ΔB), 2(ΔB)+2(RΔ), and 4(ΔΔ) channels. Data for each plotted point is from 5–7 single-channel patches. The lines are fits with the Hill equation (Eq. 1). The data from the 4(RΔ), 4(ΔB), and 2(ΔB)+2(RΔ) channels cluster together, and consequently these data were simultaneously fit to obtain the continuous line through the data. The K _d for the 4(RB) and combined 4(RΔ), 4(ΔB), and 2(ΔB)+2(RΔ) 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.

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(ΔΔ) and 2(ΔB)+2(RΔ) channels, both adjacent and diagonal subunit configurations are shown. (B) 2(RB)+2(ΔΔ) channels were obtained by coexpressing RB subunits together with ΔΔ subunits that had the Y294V pore mutation to relieve TEA_O block. Each expressed channel had one of five distinct current amplitudes in the presence of 1.5 mM TEA_O. 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 TEA_O.) The deduced subunit stoichiometry for each of the current amplitudes is shown at the right, with both possible configurations for the 2(RB)+2(ΔΔ) channel shown. The 2(RB)+2(ΔΔ) channel can be identified by a current level of ∼5 pA at + 50 mV. (C) Plot of single-channel current amplitude against the number of ΔΔ subunits, assuming that the current amplitude is proportional to the number of subunits with the pore site mutation that removes TEA_O block. (D and E) 2(ΔB)+2(RΔ) channels were obtained by coexpressing (ΔB) subunits together with RΔ subunits that had the Y294V pore mutation that relieved TEA_O block. 2(ΔB)+2(RΔ) channels were identified by a current level of ∼5 pA at + 50 mV, following the same strategy as used above.

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(ΔΔ) channel (A) and a 2(ΔB)+2(RΔ) 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. TEA_O (1.5 mM) was present for both channels. (C) Plots of Po vs. Ca²⁺ _i for the indicated channel types: 2(RB)+2(ΔΔ) channels (filled diamonds, K _d = 29.7μM, Hill coefficient = 1.35) require less Ca²⁺ _i for the same Po than 2(ΔB)+2(RΔ) channels (open circles, K _d = 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 5–7 single-channel patches.

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), K _BC = 6.3 μM, K _BO = 1.0 μM, K _RC = 7.8 μM, K _RO = 1.3 μM, K _MC = 3000 μM; K _MO = 644 μM, W = 1.19, P _max = 0.90. An assumption that the two high-affinity Ca²⁺ sites were identical gave an equally good fit (not shown) with L(V) = 2500, K _BC = K _RC = 6.9 μM, K _BO = K _RO = 1.1 μM, K _MC = 3000 μM, K _MO = 644 μM, W = 1.19. The values of n for each curve were set by the subunit composition (see text). K _MC 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(RΔ), 2(RB)+2(RΔ), 1(RB)+3(RΔ), and 4(RΔ). The continuous lines are simultaneous fits with Scheme 2 to all the data in part B, and were calculated with L(V) = 2500, K _BC = 7.18 μM, K _BO = 1.00 μM, K _RC = 110.35 μM, K _RO = 18.98 μM, K _MC = 3000 μM; K _MO = 961 μM, W = 1.63, and P _max = 0.95.

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 (A1–A3) a flexible intrasubunit interface or (B1–B3) a fixed intrasubunit interface. Transmembrane segments S0–S5 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.