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.

Armstrong, Inactivation of the sodium channel. II. Gating current experiments. 1977, Pubmed

Armstrong, Inactivation of the sodium channel. II. Gating current experiments. 1977, Pubmed
Bao, Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit. 2005, Pubmed , Xenbase
Behrens, hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family. 2000, Pubmed
Bentrop, NMR structure of the "ball-and-chain" domain of KCNMB2, the beta 2-subunit of large conductance Ca2+- and voltage-activated potassium channels. 2001, Pubmed , Xenbase
Bezanilla, Inactivation of the sodium channel. I. Sodium current experiments. 1977, Pubmed
Bezanilla, Molecular basis of gating charge immobilization in Shaker potassium channels. 1991, Pubmed , Xenbase
Brenner, Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. 2000, Pubmed
Cha, Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. 1997, Pubmed , Xenbase
Cha, Structural implications of fluorescence quenching in the Shaker K+ channel. 1998, Pubmed
Choi, Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. 1991, Pubmed
Cox, Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity. 2000, Pubmed , Xenbase
Díaz, Role of the S4 segment in a voltage-dependent calcium-sensitive potassium (hSlo) channel. 1998, Pubmed , Xenbase
Gribkoff, Maxi-K potassium channels: form, function, and modulation of a class of endogenous regulators of intracellular calcium. 2001, Pubmed
Ha, Functional effects of auxiliary beta4-subunit on rat large-conductance Ca(2+)-activated K(+) channel. 2004, Pubmed , Xenbase
Hanner, The beta subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin. 1997, Pubmed
Haug, Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part I: Aspartate 292 modulates K+ conduction by external surface charge effect. 2004, Pubmed , Xenbase
Ho, Site-directed mutagenesis by overlap extension using the polymerase chain reaction. 1989, Pubmed
Horrigan, Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. 2002, Pubmed , Xenbase
Hoshi, Biophysical and molecular mechanisms of Shaker potassium channel inactivation. 1990, Pubmed , Xenbase
Hoshi, Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. 1991, Pubmed , Xenbase
Hu, Variants of the KCNMB3 regulatory subunit of maxi BK channels affect channel inactivation. 2003, Pubmed , Xenbase
Jiang, Human and rodent MaxiK channel beta-subunit genes: cloning and characterization. 1999, Pubmed
Latorre, Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage. 2006, Pubmed
Lippiat, Properties of BK(Ca) channels formed by bicistronic expression of hSloalpha and beta1-4 subunits in HEK293 cells. 2003, Pubmed , Xenbase
Lu, MaxiK channel partners: physiological impact. 2006, Pubmed
Mannuzzu, Direct physical measure of conformational rearrangement underlying potassium channel gating. 1996, Pubmed , Xenbase
Meera, A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin. 2000, Pubmed , Xenbase
Meera, A calcium switch for the functional coupling between alpha (hslo) and beta subunits (Kv,cabeta) of maxi K channels. 1996, Pubmed
Moss, Gating and conductance properties of BK channels are modulated by the S9-S10 tail domain of the alpha subunit. A study of mSlo1 and mSlo3 wild-type and chimeric channels. 2001, Pubmed , Xenbase
Nimigean, The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states. 1999, Pubmed
Orio, New disguises for an old channel: MaxiK channel beta-subunits. 2002, Pubmed
Orio, Differential effects of beta 1 and beta 2 subunits on BK channel activity. 2005, Pubmed , Xenbase
Orio, Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel. 2006, Pubmed , Xenbase
Perozo, Gating currents in Shaker K+ channels. Implications for activation and inactivation models. 1992, Pubmed , Xenbase
Perozo, Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. 1993, Pubmed
Qian, Intra- and intersubunit cooperativity in activation of BK channels by Ca2+. 2006, Pubmed , Xenbase
Roux, Fast inactivation in Shaker K+ channels. Properties of ionic and gating currents. 1998, Pubmed , Xenbase
Salkoff, High-conductance potassium channels of the SLO family. 2006, Pubmed
Savalli, Voltage-dependent conformational changes in human Ca(2+)- and voltage-activated K(+) channel, revealed by voltage-clamp fluorometry. 2006, Pubmed , Xenbase
Schreiber, A novel calcium-sensing domain in the BK channel. 1997, Pubmed , Xenbase
Shen, Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation. 1994, Pubmed , Xenbase
Solaro, The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers. 1997, Pubmed
Stefani, Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo). 1997, Pubmed , Xenbase
Stefani, Cut-open oocyte voltage-clamp technique. 1998, Pubmed , Xenbase
Tombola, How does voltage open an ion channel? 2006, Pubmed
Toro, KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals. 2006, Pubmed
Tseng-Crank, Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain. 1996, Pubmed , Xenbase
Uebele, Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel. 2000, Pubmed
Wallner, Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca(2+)-sensitive K+ channels: an additional transmembrane region at the N terminus. 1996, Pubmed
Wallner, Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane beta-subunit homolog. 1999, Pubmed , Xenbase
Wallner, Characterization of and modulation by a beta-subunit of a human maxi KCa channel cloned from myometrium. 1995, Pubmed , Xenbase
Wang, Consequences of the stoichiometry of Slo1 alpha and auxiliary beta subunits on functional properties of large-conductance Ca2+-activated K+ channels. 2002, Pubmed , Xenbase
Wang, Mechanism of beta4 subunit modulation of BK channels. 2006, Pubmed
Wei, Calcium sensitivity of BK-type KCa channels determined by a separable domain. 1994, Pubmed
Weiger, A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels. 2000, Pubmed
Xia, Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. 1999, Pubmed , Xenbase
Xia, Rectification and rapid activation at low Ca2+ of Ca2+-activated, voltage-dependent BK currents: consequences of rapid inactivation by a novel beta subunit. 2000, Pubmed , Xenbase
Xia, Multiple regulatory sites in large-conductance calcium-activated potassium channels. 2002, Pubmed
Zarei, A novel MaxiK splice variant exhibits dominant-negative properties for surface expression. 2001, Pubmed
Zarei, Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel. 2007, Pubmed
Zeng, Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites. 2005, Pubmed , Xenbase
Zeng, BK channels with beta3a subunits generate use-dependent slow afterhyperpolarizing currents by an inactivation-coupled mechanism. 2007, Pubmed
Zeng, Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels. 2003, Pubmed