Savalli N , Kondratiev A , de Quintana SB , Toro L , Olcese R .

HL054970 NHLBI NIH HHS , R01NS043240 NINDS NIH HHS , R01 HL054970 NHLBI NIH HHS , R01 NS043240 NINDS NIH HHS

	Figure 1. Schematic topology of BKCa α and β2 subunits. (A) BKCa α subunit is formed by seven trasmembrane domains (S0–S6), an extracellular N terminus and a long intracellular C terminus, which comprises four hydrophobic domains (S7–S10). β2 subunit possesses two transmembrane domains (T1 and T2) and an N-terminus that confers the inactivating properties to the channel. (B) Detail of the S3–S4 region illustrating the position of the three residues (NRS) that have been individually substituted with cysteines and labeled with different fluorophores (see Materials and Methods).
	Figure 2. Lack of voltage-dependent fluorescence changes in the hSlo C-less background channel. Representative K+ current traces from oocytes expressing α (A) and α+β2 (C), elicited by 100-ms depolarization from −160 mV to the indicated potential. The corresponding TMRM fluorescence traces are shown in B and D. Holding potential (HP) was −90 mV. In C, the time- dependent decay of the ionic current is caused by the inactivating properties conferred to the BKCa channel by β2 subunits. Note that no fluorescence changes (ΔF) were detected in hSlo C-less. (E) Averaged G(V) curves for α (◯) and α+β2(□) and the best fits to one Boltzmann distribution are shown superimposed (see Materials and Methods). Coexpression of the β2 subunit produced ∼20 mV shift of the G(V) curve toward more negative potential (fitting parameters: α, Vhalf = 1.44 mV and z = 0.84; α+β2, Vhalf = −26.13 mV and z = 0.83). Error bars represent SEM.
	Figure 3. Conformational changes in α and α+β2 reported by PyMPO at labeling position 202. Representative K+ current traces from oocytes expressing α (A) and α+β2 (D), elicited by 1-s depolarization from −160 mV to the indicated potential (HP = −90 mV). The corresponding fluorescence traces and the best fits to a single exponential function are shown superimposed in B and E. (C) Averaged F(V) and G(V) curves for α (G(V), ◯, and F(V), •) and α+β2 (G(V), □, and F(V), ▪). Data points are fitted to a single Boltzmann distribution and normalized to the respective maxima and minima (see Materials and Methods). Note that the coexpression of β2 subunits produced a G(V) leftward shift of ∼15 mV associated to a corresponding shift of the F(V) (∼20 mV). G(V) fitting parameters are: α, Vhalf = 0.94 mV and z = 1.08; α+β2, Vhalf = −13.02 mV and z = 1.00. F(V) fitting parameters are: α, Vhalf = −43.50 mV and z = 0.78; α+β2, Vhalf = −68.69 mV and z = 0.62. (F) Kinetics of fluorescence and current activation. Both current and fluorescence activation were approximated to one exponential function. The coexpression of β2 subunits reduced the rate of current activation in all potentials range (at 40 mV, τα = 2.52 ± 0.18 ms, n = 6, and τα+β2 = 3.89 ± 0.62 ms, n = 4). A similar effect of the β2 subunits is observed on the rate of the fluorescence onset (at 40 mV, τα = 200 ± 13.24 ms, n = 6, and τα+β2 = 392.67 ± 19.83 ms, n = 4). Error bars represent SEM.
	Figure 4. Conformational changes in α and α+β2 reported by PyMPO at labeling position 201. Representative K+ current traces from oocytes expressing α (A) and α+β2 (C), elicited by 100-ms depolarization from −160 mV to the indicated potential (HP = −90 mV). The corresponding fluorescence traces and the best fits to a single exponential function are shown superimposed in B and D. (E) Averaged F(V) and G(V) curves for α (G(V), ◯, and F(V), •) and α+β2 (G(V), □, and F(V), ▪). Data points are fitted to a single Boltzmann distribution and normalized to the respective maxima and minima (see Materials and Methods). Note that coexpression of β2 subunits produced a leftward shift of the conductance curve (∼10 mV) associated to a corresponding more relevant shift of the fluorescence curve (∼30 mV). G(V) fitting parameters are: α, Vhalf = 16.73 mV and z = 0.98; α+β2, Vhalf = 3.72 mV and z = 1.06. F(V) fitting parameters are: α, Vhalf = −19.81 mV and z = 0.67; α+β2, Vhalf = −56.89 mV and z = 0.64. (F) Kinetics of fluorescence and current activation. Both current and fluorescence activation were approximated to one exponential function. The coexpression of β2 subunits reduced the rate of current activation in all potentials range (at 40 mV, τα = 2.73 ± 0.18 ms, n = 8, and τα+β2 = 5.22 ± 1.05 ms, n = 6). A similar effect of the β2 subunits is observed on the rate of the fluorescence onset (at 40 mV, τα = 3.47 ± 0.09 ms, n = 8, and τα+β2 = 17.35 ± 3.69 ms, n = 6). Error bars represent SEM.
	Figure 5. Conformational changes in α and α+β2 reported by TMRM at labeling position 200. Representative K+ current traces from oocytes expressing α (A) and α+β2 (C), elicited by 100-ms depolarization from −160 mV to the indicated potential (HP = −90 mV). The corresponding fluorescence traces and the best fits to a single exponential function are shown superimposed in B and D. (E) Averaged F(V) and G(V) curves for α (G(V), ◯, and F(V), •) and α+β2 (G(V), □, and F(V), ▪). Data points are fitted to a single Boltzmann distribution and normalized to the respective maxima and minima (see Materials and Methods). Note that coexpression of β2 subunits produced a leftward shift of the conductance curve (∼5 mV) associated to a corresponding more relevant shift of the fluorescence curve (∼20 mV). G(V) fitting parameters are: α, Vhalf = −10.31 mV and z = 0.88; α+β2, Vhalf = −18.93 mV and z = 0.97. F(V) fitting parameters are: α, Vhalf = −84.57 mV and z = 0.93; α+β2, Vhalf = −103.66 mV and z = 0.75. (F) Kinetics of fluorescence and current activation. Both current and fluorescence activation were approximated to one exponential function. The coexpression of β2 subunits reduced the rate of current activation at all potentials tested (at 40 mV, τα = 2.00 ± 0.28 ms, n = 5, and τα+β2 = 4.19 ± 0.45 ms, n = 3). Error bars represent SEM.
	Figure 6. Ca2+ dependence of the half activation potential (Vhalf) in hSlo C-less R207Q S202C. (A and B) Representative K+ current traces (unsubtracted) from excised patches of oocytes expressing hSlo α subunit elicited by 50-ms depolarizations from −160 mV to the indicated potential (HP = −90 mV). (C and D) Representative K+ current traces from excised patches of oocytes expressing hSlo α+β2 subunits elicited by 80-ms depolarizations from −240 mV to the indicated potential (HP = −90 mV). A 25-ms prepulse to −240 mV was necessary to recover channel from inactivation. (E) Scatter plot showing the dependence of Vhalf on the log[Ca2+] for α (◯) and α+β2 (□). Continuous lines are the linear regression of the experimental data, shown superimposed. The β2 subunit facilitates channel opening, significantly lowering the Vhalf in the range of [Ca2+] tested (P < 0.0001) (ANCOVA).
	Figure 7. S4 immobilization revealed by OFF fluorescence kinetics in Shaker M356C W434F. Representative gating current traces from oocytes expressing Sh-IR M356C W434F (A) or Sh M356C W434F (B) elicited by 40-ms depolarization from −90 mV to the indicated potential (HP = −90 mV). Note the slow OFF-charge return in the presence of the intact N terminus (B). The corresponding fluorescence traces and the best fits to one (C) or two (D) exponential function(s) are shown superimposed in B and D. The appearance of a second component in the exponential function fitting fluorescence deactivation reveals the immobilization of the S4 segment in the activated position (at 10 mV, for Sh-IR τ = 3.44 ± 0.38 ms, n = 5, and for Sh τfast = 2.74 ± 0.25 ms and τslow = 32.71 ± 0.20 ms, n = 5).
	Figure 8. Evidences of S4 immobilization in Shaker channel from voltage clamp fluorometry. (A) Averaged Q(V) and F(V) curves for Sh (Q(V), ◯, and F(V), •) and Sh-IR (Q(V), □, and F(V), ▪). Data points are fitted to two Boltzmann distributions and normalized to the respective maxima and minima (see Materials and Methods). Note that the inactivation process is not affecting the voltage dependence of both charge and fluorescence. The averaged parameters are as follows: in Sh-IR, for the Q(V) Vhalf1 = −56.40 ± 1.58 mV, z1 = 1.68 ± 0.06, Q1 = 22.50 ± 1.59%, Vhalf2 = −21.01 ± 1.31 mV, z2 = 1.86 ± 0.05, Q2 = 77.50 ± 1.59% and for the F(V) Vhalf1 = −44.53 ± 1.93 mV, z1 = 1.17 ± 0.08, F1 = 30.37 ± 2.62%, Vhalf2 = −20.38 ± 1.50 mV, z2 = 2.79 ± 0.19, F2 = 69.63 ± 2.62% −ΔF/F = 2.72 ± 0.59% (n = 5); in Sh, for the Q(V) Vhalf1 = −61.72 ± 2.04 mV, z1 = 1.50 ± 0.17, Q1 = 18.44 ± 2.26%, Vhalf2 = −16.11 ± 1.87 mV, z2 = 1.78 ± 0.15, Q2 = 81.56 ± 2.26% and for the F(V) Vhalf1 = −41.38 ± 2.24 mV, z1 = 1.66 ± 0.18, F1 = 34.30 ± 3.44%, Vhalf2 = −22.34 ± 0.55 mV, z2 = 3.24 ± 0.17, F2 = 65.70 ± 3.44% −ΔF/F = 1.19 ± 0.18% (n = 5). (B) Averaged time constants (n = 5) of the ON fluorescence for Shaker (◯) and Shaker-IR (•) at different membrane potentials (HP = −90mV) (fit to a monoexponential function). (C) Averaged time constants (n = 5) of the OFF fluorescence for Shaker (◯, •) and Shaker-IR (▪) at different membrane potentials (HP = −90mV). The OFF fluorescence was fitted to single exponential function for Sh-IR and to the sum of two exponential functions for Sh. In the presence of the inactivating “ball” a second, slower component appears in the fluorescence traces during repolarization. Note that the time constant of the fast component of Sh (◯) closely follows the one of Sh-IR (▪), while the second component in Sh (•) (generated by inactivated channels) is ∼10-fold slower. (D) Percentage of fast (◯) and slow (•) components of the OFF fluorescence in Sh. Error bars represent SEM. When the bars are not visible, they are inside the symbols.
	Figure 9. The β2-induced inactivation process is not interfering with BKCa S4 segment return in the resting position. Representative K+ current traces from oocytes expressing the α (A and E) and α+β2 subunit (S202C mutant) (C and G), elicited by 1-s depolarization from −160 to 80 mV, and repolarizations to −160 mV (A and C) and to −90 mV (E and G) (HP = −90 mV). The corresponding fluorescence traces and the best fits to a single exponential function of the OFF fluorescence are shown superimposed in B, D, F, and H. Note that the OFF fluorescence is well fitted to a single exponential function both in the presence and in the absence of β2 subunits. (I) Time course of fluorescence return at different membrane potentials. (L) Time constants of current inactivation (◯), best fitted to one exponential function from current peak to the end of the pulse (1-s pulses, as shown in Fig. 3 D), were compared with time constants of ON fluorescence (▴) and OFF fluorescence (▪) (during repolarization to −160 mV) when β2 is coexpressed, both best fitted to one exponential function. There is no correlation between the current inactivation kinetics and the fluorescence kinetics. Error bars represent SEM. When the bars are not visible, they are inside the symbols.
	Figure 10. Voltage dependence of the recovery from β2-induced inactivation. (A and B) Family of K+ currents from oocytes expressing α+β2 subunits (S202C mutant) elicited by a “double pulse protocol” shown above the traces (HP = −90 mV). A 700-ms inactivating pulse from −160 to 40 mV is followed by repolarizing interpulses to −160 mV (A) or to −90 mV (B) of variable duration. A 25-ms test pulse to 40 mV is applied to monitor the recovery from inactivation. C and D are an expanded scale of the ionic current during test pulses as in A and B, respectively. (E) Semilogarithmic plot of the percentage of fractional recovery calculated as the ratio between the peak current elicited by the test pulse and the peak current elicited by the inactivating pulse for the experiment shown in A and B. The fits to biexponential functions are shown superimposed. (F) Averaged time constants for the recovery from inactivation at different membrane potentials: at −160 mV, τfast = 5.24 ± 0.35 ms and τslow = 45.87 ± 5.65 ms; at −120 mV, τfast = 17.89 ± 1.34 ms and τslow = 135.96 ± 8.27 ms; at −90 mV, τfast = 43.47 ± 3.99 ms and τslow = 248.91 ± 0.23 ms (n = 4). The relative amplitudes of the two components of the exponential fits are shown in G (at −160 mV, Ampfast% = 88.23 ± 2.88% and Ampslow% = 11.77 ± 2.88%; at −120 mV, Ampfast% = 70.24 ± 3.25% and Ampslow% = 29.76 ± 2.88%; at −90 mV, Ampfast% = 57.16 ± 2.60% and Ampslow% = 42.84 ± 2.88%, n = 4). Error bars represent SEM.

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