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Figure 1. Coexpression of α+β2+γ1 subunits results in all BK channels containing both β- and γ-subunits.(a–b) Cartoons of the predicted topology of α-, β2- and γ1-subunits. The basic topology of β2- and γ1-subunits are shared with other members of their respective subunit families. (c) Schematic of deduced position of α- and β1-subunit TM segments in a BK channel complex viewed at the extracellular face17. Grey, pink and blue ellipses highlight voltage sensor domain (VSD), pore domain (PD) and β-subunit TMs, respectively. (d–g) Typical currents arising from (co)expression of BK subunits in oocytes: α alone, α+γ1, α+β2, or α+β2+γ1, respectively, at 0 (left) or 10 μM intracellular [Ca2+] (right); left and right currents were from the same patch using the same voltage protocol (depicted for each case). (h–i) Comparison among activation curves (G–V) at 0 Ca2+ or 10 μM of Ca2+, respectively, generated from sets of experiments as in panels (d–g). Notice the overlap between activation curves at 0 Ca2+ of α+γ1 and α+β2+γ1. G–V fits to single Boltzmann distributions yielded the following parameters: At 0 Ca2+, α-alone (Vh=193±6 mV, z=0.7±0.02 e0, n=5); α+β2ΔNt (Vh=182±4 mV, z=0.7±0.03 e0, n=5) ; α+γ1 (Vh=29.0±4.7 mV, z=1.3±0.08 e0, n=7); α+β2+γ1 (Vh=28.9±3.4 mV, z=0.9±0.04 e0, n=9). At 10 μM of Ca2+, α-alone (Vh =22.6±3 mV, z=1.4±0.12 e0, n=5); α+β2ΔNt (Vh=−9.2±4.4 mV, z=1.6±0.87 e0,
n=5); α+γ1 (Vh=−95.1±3.9 mV, z=1.4±0.05 e0, n=8). (j) Averages of τinact at voltages up to +180 for α+β2+γ1 at 0 Ca2+ (n=9) and α+β2 at 10 Ca2+ (n=6), respectively. All errors are s.e.m.
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Figure 2. The voltage dependence of the fraction of channels available to activate also reflects the coassembly of β2- and γ1-subunits in the same channels.(a–b) Examples of the fractional availability of channels as function of the conditioning potential from oocytes injected with α+β2 (10 μM Ca2+) or α+β2+γ1 (0 Ca2+), respectively. Currents were evoked by the indicated protocols using conditioning potentials of 100 ms duration. (c–d) Curves of activation (open symbols) and fractional availability (filled symbols) as function of voltage obtained at 10 μM of Ca2+ for α+β2 or 0 Ca2+ for α+β2+γ1 channels, respectively, are plotted together. G–V fits to single Boltzmann distributions yielded the following parameters: α+β2 SS-inactivation (Vh=−110.8±2.3 mV, z=1.81±0.11 e0, n=7) α+β2-activation (Vh=−6.7±2.4 mV, z=0.96±0.31 e0, n=6); α+β2+γ1-SS-inactivation (Vh=−113.5±5.3 mV, z=1.56±0.12 e0, n=4), α+β2+γ1-activation (also shown in Fig. 1h). All errors are s.e.m.
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Figure 3. Single α+β2+γ1 channels simultaneously exhibit the maximal γ1-induced effect and up to four β2-subunits.(a–b) Cartoons representing two distinctive assembly models: where α, white; β2, blue; γ1, red. (c–f) Representative single channels obtained from various α:β2:γ1 injection ratios (given above each set of traces). For each example, five consecutive traces of current recorded at 0 Ca2+ are shown together with the ensemble current average from 100 total traces recorded in the same conditions. Red lines: fit of a single exponential with the indicated τinact. (g–h) Grey bars represent the distribution of τinact from 31 single-channels (bin size: 2 ms). Red lines represent fits of the binned data to four- or three-component Gaussian distributions, respectively, both with the s.d. (sn) constrained such that s1≥
s2≥
s3…sn. Mean values (τn) and sum of the squares of the errors (SSQ) obtained from each fit are indicated. The other parameters of each Gaussian component, amplitude (An) and standard deviation, of the distributions are: four-component distribution (A1=2.1, s1=3.0; A2=3.5, s2=1.8; A3=5.6, s3=1.2; A4=4.3, s4=1.2) and three-component distribution (A1=2.05, s1=3.1; A2=2.4, s2=3.1; A3=3.6, s3=3.1).
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Figure 4. γ1 and β2 induced gating shifts are largely additive.(a–b) Currents arising from coexpression of α+β2-inactivation removed (β2ΔNt) (a) or α+β2ΔNt+γ1 (b) with 10 μM Ca2+. (c) Comparison of the gating shift obtained by β2 with or without the γ1 expression. β2 produces an approximately 30–35 mV shift with or without γ1. With 10 μM Ca2+, Vh for (α alone)=+22.6±3.4 mV, (α+γ1)=−95.1±3.9 mV, (α+β2ΔNt)=−9.2±4.4 mV and (α+β2ΔNt+γ1)=−134.7±8.8 mV (n=5); ΔV(β2)=31.8, ΔV(γ1)=117.7, ΔV(β2ΔNt+γ1)=157.3. All errors are s.e.m.
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