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J Gen Physiol
2002 Dec 01;1206:829-43. doi: 10.1085/jgp.20028692.
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Slo1tail domains, but not the Ca2+ bowl, are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels.
Qian X, Nimigean CM, Niu X, Moss BL, Magleby KL.
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Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.
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Figure 1. . Schematic diagram of the membrane topology of the β1 and α subunit of the BK (Slo1) channel is shown in the top panel. The core and tail region of the α subunit are indicated as well as the RCK1, RCK2, and serine proteinase (SerP) like domains. The unconserved linker between S8 and S9 connects the core and tail as well as the two RCK domains. The tail contains the Ca2+ bowl with five consecutive aspartic acid residues, implicated with high affinity Ca2+ binding. Schematic diagrams of the channels examined in this study are indicated below the topology diagrams. Note that the linker region between the S8-S9 is missing in the Slo1 core/Slo3 tail channel, and is present in the Slo1 core/Slo3 tail joined channel.
Figure 2. . The β1 subunit increases the apparent Ca2+ sensitivity of the Slo1core/Slo1 tail channels while reducing the voltage sensitivity. (A and B) Representative single-channel current traces recorded from Slo1 core/Slo1 tail channels with and without β1 subunits for 1.1, 2.5, 5.7 μM Ca2+i, as indicated, at 30 mV. (C and D) Representative macrocurrents recorded for 0 and 20 μM Ca2+i with and without the β1 subunit. For currents with 0 Ca2+i, the potential was held at −80 mV, stepped to potentials between 0 and 300 mV in increments of 20 mV, and then stepped to 40 mV to measure tail currents. For currents with 20 μM Ca2+i, the potential was held at −120 mV, stepped to potentials between −100 to 120 mV in increments of 10 mV and then stepped to −80 mV to measure tail currents. (E) G-V curves derived from tail currents of Slo1core/Slo1 tail channels with and without β1 subunits. Each point plots the mean from >5 different patches. The lines in the GV plots in this and later figures are the fitted Boltzmann function with Eq. 1. The average voltage sensitivity was 20.2 ± 1.0 mV/e-fold without the β1 subunit and 26.3 ± 2.1 mV/e-fold with the β1 (F) Plots of the V0.5 versus Ca2+i for experiments like in (E). V0.5 was determined separately for data from each of >5 different patches and then averaged.
Figure 3. . The β1 subunit had little effect on burst duration and Po of Slo1 core/Slo3 tail channels at 30 mV, while decreasing the voltage sensitivity. (A and B) Representative single-channel currents recorded from Slo1core/Slo3 tail channels with and without β1 subunits, for 0, 20, and 100 μM Ca2+i at 30 mV. (C and D) Representative macrocurrents recorded with and without β1 subunits, for either 0 or 20 μM Ca2+i. Currents were held at −100 mV and then activated by stepping to potentials between −50 to 200 mV in increments of 10 mV, with tail currents measured at −80 mV. (E) G-V curves derived from tail currents of Slo1core/Slo3 tail channels with and without β1 subunits. The average voltage sensitivity was 22.6 ± 0.4 mV/e-fold without β1 subunits and 28.8 ± 0.5 mV/e-fold with β1 subunits. (F) Plots of V0.5 for Slo1 core/Slo3 tail channels with and without β1 subunits.
Figure 4. . Comparison of the single-channel kinetics for various channel constructs with and without β1 subunits. Slo1 core/Slo1 tail channels: 2.5 μM Ca2+i. Slo1 core/Slo3 tail channels: 0 μM Ca2+i. Slo1 with D965N mutation: 20 μM Ca2+i. Average of 5–9 patches in each case, except 3 patches for burst duration and gap duration of D965N.
Figure 5. . DHS-I and estrogen activate Slo1 core/Slo3 tail channels only in the presence of the β1 subunit, indicating that the β1 subunit is functionally associated with Slo1 core/Slo3 tail channels. (A and B) Representative single-channel currents from Slo1core/Slo3 tail channels with and without β1 subunits and 100 nM intracellular DHS-I, as indicated. (C and D) Representative single-channel currents from Slo1core/Slo3 tail channels with and without β1 subunits and 10 μM extracellular 17β-estradiol, as indicated.
Figure 6. . The β1 subunit greatly increases burst duration and Po, and leftward shifts V0.5 after the Ca2+ bowl mutation D965N to the Slo1 channel. (A) Representative single-channel currents recorded from Slo1 channels, and Slo1 channels with the Ca2+ bowl mutation D965N without, and with β1 subunits. 20 μM Ca2+i, 30 mV. (B) Representative macrocurrents recorded from the D965N mutant Slo1 channels with and without β1 subunits. The potential was held at −120 mV, stepped to potentials between −100 to 200 mV in increments of 10 mV, and then stepped to −80 mV to measure tail currents. (C) G-V curves derived from tail currents of Slo1 channels, D965N mutant Slo1 channels, and D965N mutant Slo1 channels with β1 subunits (20 μM Ca2+i). The average voltage sensitivity was 19.1 ± 0.6 mV/e-fold without the β1 subunit and 23.1 ± 1.2 mV/e-fold with the β1 (n = 5 patches in each case except 3 patches for Slo1). (D) Plots of V0.5 for Slo1 channels, and D965N mutant Slo1 channels without and with β1 subunits.
Figure 7. . The β1 subunit greatly increases burst duration and Po after a Ca2+ bowl mutation that deletes D965 and D966 of Slo1. (A) Representative single-channel currents recorded from Slo1 channels, and Slo1 channels with the Ca2+ bowl mutation that deletes D965 and D966 without and with β1 subunits. 8 μM Ca2+i, 50 mV. (B) Plots of mean burst duration versus Ca2+i for three mutant channels and four mutant channels with the β1 subunit.
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