Zhang G , Geng Y , Jin Y , Shi J , McFarland K , Magleby KL , Salkoff L , Cui J .

R01 GM114694 NIGMS NIH HHS , R01 HL070393 NHLBI NIH HHS , R01 NS092570 NINDS NIH HHS

	(Scheme 1).
	Figure 1. Effects of gating ring removal on gating charge movements of BK channels. (A) Gating currents of WT and Core-MT mSlo1 channels. Voltage pulses were from −30 to 300 mV (WT) or from −80 to 300 mV (Core-MT) with 20-mV increments. (B) Normalized gating charge-voltage (Q-V) curve of on-gating currents. The smooth curves are fits to the Boltzmann function with a V1/2 and slope factor (b in Eq. 1) of 159.1 ± 6.5 mV and 49.0 ± 5.9 mV for WT and 138.0 ± 3.1 mV and 51.3 ± 2.8 mV for Core-MT. V1/2 of the on-gating current gives the half-activation voltage at the closed state, Vhc. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 2. Removal of gating ring alters voltage-dependent opening of BK channels. (A) Macroscopic currents of WT and the Core-MT channels. The currents were elicited in 0 [Ca2+]i by voltage pulses from −30 to 250 mV with 20-mV increments. The voltages before and after the pulses were −50 and −80 mV, respectively. (B) Conductance-voltage (G-V) curves for WT and Core-MT channels in 0 [Ca2+]i. Solid lines are fits to the Boltzmann relation (see Materials and methods) with V1/2 and slope factor for WT of 184.5 ± 3.1 mV and 20.5 ± 2.8 mV and for Core-MT of 244.2 ± 3.8 mV and 27.6 ± 3.8 mV. (C) Currents of E321A/E324A WT and E321A/E324A Core-MT channels in 0 [Ca2+]i. (D) G-V curves for E321A/E324A WT and E321A/E324A Core-MT channels in 0 [Ca2+]i. Solid lines are fits to the Boltzmann relation with V1/2 and slope factor for E321A/E324A WT of 81.3 ± 3.2 mV and 19.5 ± 2.8 mV and for E321A/E324A Core-MT of 154.4 ± 3.2 mV and 28.9 ± 2.9 mV. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 3. Deletion of gating ring alters open probability at voltages <100 mV. (A) Removing the CTD from mSlo1 reduces single-channel conductance. Representative single-channel current recordings from WT (top three panels) and Core-MT (bottom three panels) at the indicated voltages obtained from inside-out patches in symmetrical 160 mM KCl. Channel openings are upwards. (B) Plots of i-V single-channel current amplitudes. The lines are cubic spline fits constrained to pass through the origin. (C) Single-channel current records at the indicated voltages from inside-out patches for WT, E321A/E324A mutated WT, and E321A/E324A mutated Core-MT as indicated. Channel openings are upwards. The activity of E321A/E324A mutated channels could wander somewhat, but much less pronounced than observed for WT dSlo1 channels (Silberberg et al., 1996). (D) PO increases with depolarization for the channels as indicated. The solid line is a fit to the Boltzmann function, with V1/2 of 186.2 ± 5.4 mV, a slope factor of 21.4 ± 4.6 mV, and a POMax of 0.94 for WT; V1/2 of 79.1 ± 4.8 mV, a slope factor of 19.3 ± 3.2 mV/e, and a POMax of 0.94 for E321A/E324A mutated WT; and V1/2 of 182.0 ± 3.2 mV, a slope factor of 25.6 ± 2.6 mV, and a POMax of 0.27 for E321A/E324A mutated Core-MT. The number of patches for each voltage and channel type ranged from three to six. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 4. PO-V relations of WT and mutated channels. (A) Unitary channel openings at −140 mV from membrane patches containing multiple channels. (B) PO/POMax-V relations at 0 [Ca2+]i. (C) Plot of PO versus voltage for WT and Core-MT channels. The smooth curves are fits to the extended HCA model with POMax = 0.27 for Core-MT and POMax = 0.94 for WT. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 5. Plots of PO versus voltage for WT and Core-MT channels. (A–C) The Core-MT data have been scaled assuming that POMax for Core-MT channels is 0.99, 0.1, and 0.01, as indicated in A–C, respectively. POMax was always 0.94 for WT channels. The smooth curves are fits to the extended HCA model with the parameters in Table 1. The voltage sensitivity of PO versus voltage (slope) of the scaled Core-MT data was always less than for the WT data, indicating that the observation of decreased coupling for Core-MT was independent of the assumed value for POMax. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 6. β1 subunit modulates voltage-dependent activation. (A) Macroscopic current traces were elicited in 0 [Ca2+]i by voltage pulses from −30 to 250 mV with 20-mV increments. The voltages before and after the pulses were −50 and −80 mV, respectively. (B) G-V curves for WT + hβ1 and Core-MT + hβ1 channels in 0 [Ca2+]i. Solid lines are fits to the Boltzmann equation with V1/2 and slope factor for WT + hβ1 of 205.0 ± 4.7 mV and 32.6 ± 4.8 mV and for Core-MT + hβ1 of 262.8 ± 6.2 mV and 32.2 ± 6.1 mV. (C) Gating current traces for WT + hβ1 and Core-MT + hβ1. Voltage pulses were from −30 to 300 mV (WT + hβ1) or from −80 to 300 mV (Core-MT + hβ1) in 20-mV increments. (D) Normalized Q-V relations of on-gating currents. The smooth curves are fits to the Boltzmann equation with V1/2 and slope factor 88.8 ± 6.8 mV and 40.1 ± 5.2 mV for WT + hβ1 and 87.6 ± 5.0 mV and 53.3 ± 4.3 mV for Core-MT + hβ1. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.
	Figure 7. β2 subunit modulates voltage-dependent activation. (A) Macroscopic current traces were elicited in 0 [Ca2+]i by voltage pulses from −30 to 250 mV with 20-mV increments. The voltages before and after the pulses were −50 and −80 mV, respectively. (B) G-V relations for WT + hβ2 and Core-MT + hβ2 in 0 [Ca2+]i. Solid lines are fits to the Boltzmann equation with V1/2 and slope factor for WT + hβ2 of 181.3 ± 3.4 mV and 24.9 ± 3.1 mV and for Core-MT + hβ2 of 244.3 ± 3.0 mV and 22.0 ± 3.1 mV. (C) Gating current traces for WT + hβ2 and Core-MT + hβ2. Voltage pulses were from −30 to 300 mV for WT + hβ2 and from −80 to 300 mV for Core-MT + hβ2 in 20-mV increments. (D) Normalized Q-V relations of on-gating currents. The smooth curves are fits to the Boltzmann equation with V1/2 and slope factor 94.2 ± 6.1 mV and 46.1 ± 5.1 mV for WT + hβ2 and 116.2 ± 6.1 mV and 44.9 ± 5.4 mV for Core-MT + hβ2. The data points represent the mean ± SEM; n ≥ 4 for all figures unless specified otherwise.

Adams, Intracellular Ca2+ activates a fast voltage-sensitive K+ current in vertebrate sympathetic neurones. 1982, Pubmed

Adams, Intracellular Ca2+ activates a fast voltage-sensitive K+ current in vertebrate sympathetic neurones. 1982, Pubmed
Adelman, Calcium-activated potassium channels expressed from cloned complementary DNAs. 1992, Pubmed , Xenbase
Alvarez, Counting channels: a tutorial guide on ion channel fluctuation analysis. 2002, Pubmed
Atkinson, A component of calcium-activated potassium channels encoded by the Drosophila slo locus. 1991, Pubmed
Bao, Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity. 2002, Pubmed , Xenbase
Bao, Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit. 2005, Pubmed , Xenbase
Barrett, Properties of single calcium-activated potassium channels in cultured rat muscle. 1982, Pubmed
Behrens, hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family. 2000, Pubmed
Braun, Voltage-gated ion channels in human pancreatic beta-cells: electrophysiological characterization and role in insulin secretion. 2008, Pubmed
Brayden, Regulation of arterial tone by activation of calcium-dependent potassium channels. 1992, Pubmed
Brelidze, A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification. 2003, Pubmed , Xenbase
Brelidze, Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages. 2004, Pubmed , Xenbase
Budelli, Properties of Slo1 K+ channels with and without the gating ring. 2013, Pubmed , Xenbase
Butler, mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels. 1993, Pubmed , Xenbase
Cox, Allosteric gating of a large conductance Ca-activated K+ channel. 1997, Pubmed , Xenbase
Cui, Allosteric linkage between voltage and Ca(2+)-dependent activation of BK-type mslo1 K(+) channels. 2000, Pubmed
Cui, Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels. 1997, Pubmed , Xenbase
Ferguson, Opening and closing transitions for BK channels often occur in two steps via sojourns through a brief lifetime subconductance state. 1993, Pubmed
Hite, Cryo-electron microscopy structure of the Slo2.2 Na(+)-activated K(+) channel. 2015, Pubmed
Hite, Structural basis for gating the high-conductance Ca2+-activated K+ channel. 2017, Pubmed
Horrigan, Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. 2002, Pubmed , Xenbase
Horrigan, Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+). 1999, Pubmed , Xenbase
Hu, Participation of the S4 voltage sensor in the Mg2+-dependent activation of large conductance (BK) K+ channels. 2003, Pubmed
Hudspeth, Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bull-frog, Rana catesbeiana. 1988, Pubmed
Hudspeth, A model for electrical resonance and frequency tuning in saccular hair cells of the bull-frog, Rana catesbeiana. 1988, Pubmed
Lancaster, Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. 1987, Pubmed
Latorre, Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. 1982, Pubmed
Lee, Modulation of BK channel gating by the ß2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1. 2010, Pubmed , Xenbase
Lee, {beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia. 2009, Pubmed , Xenbase
Li, Nuclear BK channels regulate gene expression via the control of nuclear calcium signaling. 2014, Pubmed
Lingle, Empirical considerations regarding the use of ensemble-variance analysis of macroscopic currents. 2006, Pubmed
Long, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. 2005, Pubmed
Marty, Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. 1981, Pubmed
Meera, A calcium switch for the functional coupling between alpha (hslo) and beta subunits (KV,Ca beta) of maxi K channels. 1996, Pubmed , Xenbase
Meera, Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus. 1997, Pubmed
Meredith, BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output. 2006, Pubmed
Nimigean, Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism. 2000, Pubmed
Niu, Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca(2+)-activated potassium channel. 2004, Pubmed , Xenbase
Orio, New disguises for an old channel: MaxiK channel beta-subunits. 2002, Pubmed
Pallotta, Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. 1981, Pubmed
Petersen, Calcium-activated potassium channels and their role in secretion. , Pubmed
Robitaille, Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. 1993, Pubmed
Rothberg, Kinetic structure of large-conductance Ca2+-activated K+ channels suggests that the gating includes transitions through intermediate or secondary states. A mechanism for flickers. 1998, Pubmed
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
Shi, Mechanism of magnesium activation of calcium-activated potassium channels. 2002, Pubmed
Silberberg, Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes. 1996, Pubmed , Xenbase
Sun, The interface between membrane-spanning and cytosolic domains in Ca²+-dependent K+ channels is involved in β subunit modulation of gating. 2013, Pubmed , Xenbase
Sweet, Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage. 2008, Pubmed
Tao, Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. 2017, Pubmed
Tseng-Crank, Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain. 1996, Pubmed , Xenbase
Wallner, Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane beta-subunit homolog. 1999, Pubmed , Xenbase
Wei, Calcium sensitivity of BK-type KCa channels determined by a separable domain. 1994, Pubmed
Wellman, Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels. 2003, Pubmed
Wu, A kinetic description of the calcium-activated potassium channel and its application to electrical tuning of hair cells. 1995, Pubmed
Wu, Structure of the gating ring from the human large-conductance Ca(2+)-gated K(+) channel. 2010, Pubmed
Xia, Multiple regulatory sites in large-conductance calcium-activated potassium channels. 2002, Pubmed
Xia, Ligand-dependent activation of Slo family channels is defined by interchangeable cytosolic domains. 2004, Pubmed , Xenbase
Xia, Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues. 2003, Pubmed , Xenbase
Yang, BK channels: multiple sensors, one activation gate. 2015, Pubmed
Yang, Interaction between residues in the Mg2+-binding site regulates BK channel activation. 2013, Pubmed , Xenbase
Yang, An epilepsy/dyskinesia-associated mutation enhances BK channel activation by potentiating Ca2+ sensing. 2010, Pubmed , Xenbase
Yang, Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1 domains. 2008, Pubmed
Yuan, Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution. 2010, Pubmed
Yuan, Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. 2011, Pubmed
Zhang, A charged residue in S4 regulates coupling among the activation gate, voltage, and Ca2+ sensors in BK channels. 2014, Pubmed , Xenbase
Zhang, Ion sensing in the RCK1 domain of BK channels. 2010, Pubmed , Xenbase