Horrigan FT , Ma Z .

NS42901 NINDS NIH HHS , R01 NS042901 NINDS NIH HHS

	Figure 1. Proposed pathways of sensor/gate interaction for different channels. (A) Kv1.2, where the charged S4 segment (light blue) in the voltage sensor is proposed to interact with the S6 activation gate (dark blue) in the pore via the S4–S5 linker (arrows). The outlines of the S1–S4 voltage sensor domain and S5–S6 pore domain for two subunits from the Kv1.2 crystal structure (Long et al., 2005a) are shown in dark and light gray, respectively, and are truncated at S1 (protein data bank code 2A79). (B) MthK, where Ca2+ binding to the cytosolic domain is proposed to cause a conformational change that pulls open the gate through short peptide linkers (dashed lines) that are unresolved in the crystal structure (Jiang et al., 2002) (protein data bank code 1LNQ). The attachment points of the linker on the cytosolic domain (in foreground and background) have been altered to better illustrate that linkers are pulled away from the pore axis. (C) A hypothetical BK channel structure formed by appending the Kv1.2 voltage sensors to MthK shows how Ca2+ and voltage may both interact with the gate to have independent effects on activation. The position of the voltage sensors was determined by superimposing the pore domains of Kv1.2 (residues 341–406) and MthK (residues 26–91) in PyMol (http://www.pymol.org). (D) The hypothetical BK channel structure also illustrates the possibility that the large cytosolic domain may interact directly with voltage sensors.
	Figure 2. Effects of Mg2+ on steady-state activation. (A) Effect of 10 mM Mg2+ on macroscopic IK evoked from mSlo1 channels in response to 50-ms pulses to different voltages in 0 Ca2+ from a holding potential of −80 mV. Mg2+ decreases outward current at high voltages owing to rapid pore block. However tail currents indicate an increase in PO and slowing of channel deactivation. (B) Mean normalized GK-V relations (Po = GK/GKmax) determined from tail currents in 10 mM Mg2+(O), 2 μM Ca2+(♦) or the absence of divalent cations (control, •). (C) Mean log(PO)-V relations corresponding to B achieve a weakly voltage-dependent limiting slope at extreme negative voltages, indicating that voltage sensors are in the resting state. Dotted lines are exponential fits to control and Ca2+ data with partial charge zL = 0.3e (Ma et al., 2006). (D) Voltage sensor mutations R207Q (▵, ▴) and R167E (□, ▪) shift both the voltage dependence of activation and Mg2+ sensitivity (0 Mg: filled symbols; 10 mM Mg2+: open symbols) relative to the WT (O, •), implying that Mg2+ action depends on voltage sensor activation and is not intrinsically voltage dependent. (E) Voltage-gating mechanism (Scheme 1). Channels undergo a closed–open (C-O) conformational change that is allosterically regulated by four independent and identical voltage sensors that can undergo a resting-activated (R-A) transition. L and J are voltage-dependent equilibrium constants (L = L0exp(zLV/kT), J = J0exp(zJV/kT) = exp(zJ[V − VhC]/kT)) and D is an allosteric factor. (F) Scheme 2 includes a ligand-binding transition (X-X·M2+) in each subunit that allosterically regulates channel opening and voltage sensor activation as described by factors C and E, respectively. Solid curves in B and C represent a fit of this model to Ca2+-dependent activation in 0–100 μM Ca2+ (Horrigan and Aldrich, 2002) (VhC = 156 mV, zJ = 0.58e, L0 = 1.06 × 10−6, zL = 0.3e, KD(Ca2+) = 11 μM, C = 8, D = 24.4, E = 2.4). The same model with identical values of VhC, zJ, L0, zL, D, and E can fit the Mg2+ data (dotted curves) if Mg2+ binding is assumed to be voltage dependent (i.e., KD(Mg2+) = KD(0)exp(−Vγ2e/kT)) (KD(0) = 9.35 mM, γ = 0.2, C = 1.66) (G) Scheme 3 assumes that ligand binding allosterically regulates voltage/sensor gate coupling and voltage sensor activation as described by allosteric factors F and E, respectively.
	Figure 3. Effects of Mg2+ on gating current were measured in the absence of permeant ions and presence of extracellular tetraethyl ammonium (TEA) to block the pore. (A) Ig evoked by 0.5-ms pulses to +200 mV in the presence (dotted traces) or absence (solid traces) of Mg2+, from two different patches testing the effects 10 or 100 mM Mg2+. (B) Mean QC-V relations in 0 (•), 10 (▵), and 100 (♦) mM Mg2+ are fit by Boltzmann functions with identical equivalent charge (zJ = 0.58e) (Horrigan and Aldrich, 2002) and were normalized by the fit amplitude (QcMax). QC represents the charge distribution for closed channels, determined by fitting the initial 50–100-μs decay of IgON with an exponential function with time constant τgFast and integrating the area under the rising phase and fit (Horrigan and Aldrich, 1999). (C) τgFast-V relations for the patch in A are shifted by −21 mV in 10 mM Mg2+. Relations are fit by functions of the form τgFast = [α(V) + β(V)]−1 where α and β are forward and backward rate constants for voltage sensor activation (0 Mg: α(0) = 58600 s−1, β(0) = 1930 s−1; 10 Mg: α(0) = 45400 s−1, β(0) = 2350 s−1) (zα = 0.3 e, zβ = −0.2e). Representative Cg-V relations obtained with admittance analysis for (D) WT or (E) E374A/E399N channels in the presence (black curves) and absence (gray curves) of 10 or 100 mM Mg2+ are fit by the derivative of a Boltzmann function with respect to voltage (dotted curves) with identical amplitude and charge (zJ = 0.58e). Shifts along the voltage axis indicate the change in VhC (ΔVhC). Arrows indicate the upper voltage limit of the fit that was determined over a range where most channels are closed (Po < 0.1). (F) ΔVhC = [VhC(Mg2+) − VhC(0 Mg2+)] for WT (♦, n = 6) and E374A/E399N (⋄, n = 4) determined from Cg-V relations.
	Figure 4. Mg2+ facilitates opening in a voltage-independent manner when voltage sensors are constitutively activated. (A) Mean log(PO)-V relations for WT (•), R207Q (▴), and R210C (▾) in 0 Mg2+. Arrows indicate the voltage range where voltage sensors of WT, R207Q, and R210C are fully activated (see text). Dotted curve represents a Boltzmann fit to R210C with partial charge z = 0.46e that describes the weak voltage dependence of channel opening in both mutants when voltage sensors are fully activated (Ma et al., 2006). (B) Mean log(PO)-V relations for R207Q show that 10 mM Mg2+ (▵) increases PO even when voltage sensors are fully activated. Dotted Boltzmann fits from −20 to +240 mV have identical charge (0.46e) but V0.5 is shifted by −91.6 mV in Mg2+, consistent with a 8.2-fold increase in the C-O equilibrium constant. (C) Mean log(PO)-V relations for R210C show that 10 mM Mg2+(▿) increases the C-O equilibrium constant in a voltage-independent manner when voltage sensors are constitutively activated. Boltzmann fits have identical charge (0.46e) but V0.5 is shifted by −122 mV in Mg2+, consistent with a 16.6-fold increase in the C-O equilibrium constant and a twofold increase in the allosteric coupling factor D.
	Figure 5. Effects of Mg2+ on different binding sites were determined by measuring PO for WT and mutant channels in 0–100 mM Mg2+ (0, blue circle; 2, □; 5, ▿; 10, blue triangle; 21, ; 100, blue diamond, mM Mg2+). (A) Normalized mean GK-V relations for the WT in 0–100 mM Mg2+. (B) Mean log(PO)-V relations for WT in 0, 10, and 100 mM Mg2+ reveal that 100 Mg2+ increases PO at extreme negative voltages. These plots represent the same data as in A but were averaged in 20 mV rather than 10-mV bins to match the voltage interval for low PO measurements. Dotted lines are exponential fits to PO at extreme negative voltages in 0 and 100 Mg2+ with partial charge zL = 0.3e. (C) Mean log(PO)-V relations in 0, 10, and 100 mM Mg2+ for E374A/E399N show that mutation of the putative Mg2+ site in RCK1 almost abolishes effects of 10 mM Mg2+ but leaves the PO increase by 100 Mg2+ (dotted lines) intact. (D) Mean log(PO)-V relations in 0, 10, and 100 mM Mg2+ for R167E achieve a limiting slope at more positive voltages than the WT, confirming that PO is increased by 100 Mg2+ when voltage sensors are in the resting state (dotted lines). Inset graph plots the ratio of NPO in the presence and absence of Mg2+ at −80 mV from a larger set of experiments. (E) Mean qa-V relations for the WT in 0, 10, and 100 mM Mg2+ were derived from the slope of log(PO)-V in B (see text). Solid curves are estimates of the QO-V relation determined by fitting the foot of the qa-V relation (to ∼80% of peak qa) with a function qa = zL + 4zJ [1 + exp([VhO − V]zJ/kT)]−1 where zL = 0.3e, zJ = 0.58e, and VhO = 26.5 ± 2.1, −33.6 ± 2.4, −65.6 ± 3.7 mV in 0, 10, and 100 mM Mg2+, respectively. (F) Plot of ΔΔVCO = ΔVhC − ΔVhO for 10 and 100 mM Mg2+ in WT (blue circle) and E374A/E399N (red square) channels where ΔVhO = [VhO(Mg2+) − VhO(0 Mg2+)] was estimated from the qa-V relation as in E and ΔVhC was determined from gating current (Fig. 3 F). Nonzero values of ΔΔVCO indicate that Mg2+ alters voltage sensor/gate coupling in the WT. However, effects on coupling are abolished by the E374A/E399N mutation. (G) Alternative representation of Scheme 2 (Fig. 2 F) shows the four states defined by the voltage sensor and gate conformation in each subunit and the equilibrium constants defined by Scheme 1 (Fig. 2 E). Boxes denote the states stabilized by ligand binding, as defined by allosteric factors C and E. (H) In Scheme 3, the allosteric factor F defines a stabilization of the OA state that alters voltage sensor/gate coupling (allosteric factor D). Dotted curves in A–E represent simultaneous fits to the Po-V, log(PO)-V, and qa-V relations in 0, 10, and 100 mM Mg2+ using Scheme 1 (Fig. 2 E) (Table I parameters WTA, E374A/E399N). Solid black curves in A and B represent an alternative fit to the WT data (WTB parameters). Red curves in A represent fits to a two-site model (Eq. 2) that assumes each subunit in the WT contains a very low affinity and an RCK1 binding site described by Schemes 2 and 3, respectively (Table II parameters).
	Figure 6. Mg2+ affects voltage sensor deactivation when channels are open. (A) Ig evoked by pulses to +200 mV of different duration (0.05–20 ms) in 0 and 10 mM Mg2+. (B) Normalized OFF currents from A decay more slowly as pulse duration increases, especially in the presence of Mg2+. Red traces represent 0.05 and 0.1-ms pulses. (C) OFF kinetics at −80 mV following brief (closed) or prolonged (open) pulses are compared by plotting the quantity Q = QOFF(t) − QOFFSS on a log scale vs. time where QOFF is the integral of IgOFF and QOFFSS is the steady-state value of QOFF(t). The “closed” traces, representing the average of 0.05 and 0.1-ms records, have an exponential time course with almost identical kinetics (τFast) in 0 Mg2+ (15.5 μs) or 10 mM Mg2+ (16.3 μs). The “open” traces, representing the average of 10, 15, and 20-ms records, were fit by triple exponential functions (0 Mg2+: qFast = 6.2 fC, τFast = 15.5 μs, qMED = 24.9 fC, τMED= 54.1 μs, qSLOW = 11.2 fC, τSLOW = 500 μs; 10 Mg2+: qFast = 2.6 fC, τFast = 16.3 μs, qMED = 10.6 fC, τMED = 127 μs, qSLOW = 39.1 fC, τSLOW = 756 μs). The Med and Slow components of the fits are shown as dotted and solid lines respectively. (D) Scheme 1* depicts the five closed (Ci) and open (Oi) states defined by Scheme 1, where i represents the number of activated voltage sensors (i = 0–4). δi and γi are forward and reverse rate constants for channel opening while α and β are rates for voltage sensor activation when channels are closed, and f = . Colored arrows represent different pathways of deactivation and QOFF components for open channels in 0 Mg2+ (top) or 10 mM Mg2+ (bottom) at −80 mV. In 10 mM Mg2+, channels can close from open states other than O0 because Mg2+ allows voltage sensors in open channels to remain activated at −80 mV (QO, Fig. 5 E). The figure illustrates one possible pathway, through the O2 state. After channels close, voltage sensors can completely deactivate in 10 mM Mg2+ (QC, Fig. 3 B). This charge movement is limited by the closing rate and therefore contributes to the Slow component of QOFF. (E) The amplitude of the three OFF components (Fast, ▴; Med, light blue square; Slow, pink circle) for the experiment in A are plotted vs. pulse duration and fit by exponential functions (0 Mg2+, τ = 2.8 ms; 10 Mg2+, τ = 2.6 ms).
	Figure 7. Effects of Mg2+ on IK kinetics. (A) Mean log(τK)-V relations in 0 (•), 10 (▵), and 100 (♦) mM Mg2+. Dotted lines are fits to the limiting slope of τK between −380 and −240 mV with an exponential function with partial charge −0.135e. Solid curves are fits to Scheme 1* (Fig. 6 D) using parameters in Table III. (B) τK-V relations from A were normalized to the 0 Mg2+ relation based on the exponential fits. (C) Open and closed dwell-time histograms for R167E at −80 mV in 0 Mg2+ from a patch containing ∼10,000 channels. Dwell time histograms were log-binned and are fit by single exponential functions, consistent with a two-state closed–open transition when voltage sensors are in the resting state. (D) NPO at −80 mV measured from steady-state recordings of 20-30 s total duration in the same patch as C as [Mg2+] was changed in sequence from 100 mM to 0, 10 mM, and back to 0 Mg2+. The data were acquired in 5-s traces and error bars represent the standard error of measurements from individual traces. (E) Mean open and closed times corresponding to D. Dotted lines in D and E indicate the mean value in 0 Mg2+.
	Figure 8. Voltage sensor/gate coupling and the mechanism of Mg2+ action. (A) Multiple pathways of voltage sensor/gate coupling may exist in BK channels. Because the cytosolic domain is connected to the gate via the RCK1–S6 linker (dashed line), interactions between cytosolic and voltage sensor domains could provide a coupling pathway (curved arrow) that is independent of that within the transmembrane domain (straight arrow). (B) A speculative model of Mg2+-dependent activation, based on an electrostatic mechanism of interaction between Mg2+ in the putative RCK1 binding site and R213 in S4 (Yang et al., 2007). The BK channel is shown in the four states defined in each subunit by the movement of the voltage sensor from resting (R) to activated (A) and by the closed (C) to open (O) conformational change. Channel opening is accompanied by an expansion of the cytosolic domain that alters the position of Mg2+ (in its binding site) relative to the transmembrane domain. The Mg2+ binding site (green semicircle) in the RCK1 domain includes coordinating residues E374 and E399 and nearby Q397 (Yang et al., 2006). Voltage sensor activation moves R213 relative to the cytosolic domain. In this way the distance and therefore electrostatic repulsion between Mg2+ and R213 (indicated qualitatively by the thickness of red boxes surrounding each state) depends on the conformation of both voltage sensor and gate, such that voltage sensor/gate coupling is enhanced. To account for the observed effects of Mg2+ on both opening and voltage sensor activation requires that the OA state be stabilized (see text). This is accomplished in the model by maintaining a similar distance between Mg2+ and R213 in all states except OA, such that all states are destabilized relative to OA. To account for a small effect of Mg2+ on voltage sensor activation when channels are closed we also assume interaction in the CA state is slightly weaker than the CR state, owing to a small outward movement of R213 upon voltage sensor activation, consistent with the small contribution of R213 to gating charge (Ma et al., 2006).

Armstrong, Charge movement associated with the opening and closing of the activation gates of the Na channels. 1974, Pubmed

Armstrong, Charge movement associated with the opening and closing of the activation gates of the Na channels. 1974, Pubmed
Avdonin, Stimulatory action of internal protons on Slo1 BK channels. 2003, Pubmed
Brayden, Regulation of arterial tone by activation of calcium-dependent potassium channels. 1992, Pubmed
Butler, mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels. 1993, Pubmed , Xenbase
Campos, Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel. 2007, 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
Diaz, Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle. 1996, Pubmed , Xenbase
Faber, Calcium-activated potassium channels: multiple contributions to neuronal function. 2003, Pubmed
Ferguson, Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block. 1991, Pubmed
Gupta, NMR studies of intracellular metal ions in intact cells and tissues. 1984, Pubmed
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, Pubmed
Horrigan, Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+). 1999, Pubmed
Horrigan, Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+). 1999, Pubmed , Xenbase
Horrigan, Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. 2002, Pubmed , Xenbase
Horrigan, Heme regulates allosteric activation of the Slo1 BK channel. 2005, Pubmed
Hu, Effects of multiple metal binding sites on calcium and magnesium-dependent activation of BK channels. 2006, Pubmed , Xenbase
Hu, Participation of the S4 voltage sensor in the Mg2+-dependent activation of large conductance (BK) K+ channels. 2003, Pubmed
Jiang, Human and rodent MaxiK channel beta-subunit genes: cloning and characterization. 1999, Pubmed
Jiang, Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel. 2001, Pubmed , Xenbase
Jiang, Crystal structure and mechanism of a calcium-gated potassium channel. 2002, Pubmed
Lingle, Mg2+-dependent regulation of BK channels: importance of electrostatics. 2008, Pubmed
Long, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. 2005, Pubmed
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Lu, Coupling between voltage sensors and activation gate in voltage-gated K+ channels. 2002, Pubmed , Xenbase
Ma, Role of charged residues in the S1-S4 voltage sensor of BK channels. 2006, Pubmed , Xenbase
Neyton, A Ba2+ chelator suppresses long shut events in fully activated high-conductance Ca(2+)-dependent K+ channels. 1996, 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
Pathak, Closing in on the resting state of the Shaker K(+) channel. 2007, Pubmed
Patton, Some precautions in using chelators to buffer metals in biological solutions. 2004, Pubmed
Romani, Regulation of cell magnesium. 1992, Pubmed
Ruta, Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. 2005, Pubmed
Shi, Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels. 2001, Pubmed , Xenbase
Shi, Mechanism of magnesium activation of calcium-activated potassium channels. 2002, Pubmed
Stefani, Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo). 1997, Pubmed , Xenbase
Tang, Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels. 2003, Pubmed
Tsien, Measurement of cytosolic free Ca2+ with quin2. 1989, Pubmed
Vergara, Calcium-activated potassium channels. 1998, Pubmed
Wellman, Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels. 2003, Pubmed
Xia, Multiple regulatory sites in large-conductance calcium-activated potassium channels. 2002, Pubmed
Yang, Tuning magnesium sensitivity of BK channels by mutations. 2006, Pubmed , Xenbase
Yang, Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels. 2007, Pubmed
Ye, Crystal structures of a ligand-free MthK gating ring: insights into the ligand gating mechanism of K+ channels. 2006, Pubmed
Zhang, Cysteine modification alters voltage- and Ca(2+)-dependent gating of large conductance (BK) potassium channels. 2005, Pubmed
Zhang, Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site. 2001, Pubmed , Xenbase