XB-ART-55077
Elife
2018 Jul 02;7. doi: 10.7554/eLife.38060.
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BK channel inhibition by strong extracellular acidification.
Zhou Y
,
Xia XM
,
Lingle CJ
.
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Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.
???displayArticle.pubmedLink??? 29963986
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???displayArticle.grants??? [+]
GM118114 National Institute of General Medical Sciences, R35 GM118114 NIGMS NIH HHS , GM118114 NIGMS NIH HHS
Genes referenced: cav1 kcnma1 nav1 pgd
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Figure 1. BK channel activation is strongly inhibited when pHO is lower than 5.(A) Macroscopic BK currents recorded from an outside-out patch perfused with extracellular solutions at pH 7 (left), pH 4 (middle), and back to pH 7. Currents were evoked by steps from −160 to +160 mV at pHO 7 and by steps from −160 mV to +200 mV at pHO 4 (20 mV increments). The pre- and post-test pulse voltages were −140 and −120 mV, respectively. 300 μM Ca2+ was included in pipette (intracellular) solution. The red traces were evoked by +60 mV pulses to highlight the much larger fractional reduction in outward current at this voltage compared to that at +160 mV (blue traces). (B) G-V curves of BK channel generated from tail currents recorded at various pHOs with values normalized to the maximum value at pHO 7. Physiologically relevant voltage range (±60 mV) is marked by two vertical dotted lines here and in panel D. Boltzmann fit results (solid lines) are Gmax = 1.06 ± 0.01, z = 0.97 ± 0.03 e, Vh = −48.8 ± 1.0 mV (pH 9), Gmax = 0.95 ± 0.01, z = 1.00 ± 0.04 e, Vh = −32.4 ± 1.2 mV (pH 7), Gmax = 0.80 ± 0.01, z = 0.97 ± 0.06 e, Vh = −11.7 ± 1.8 mV (pH 6), Gmax = 0.66 ± 0.01, z = 0.86 ± 0.05 e, Vh =+34.8 ± 2.3 mV (pH 5), Gmax = 0.46 ± 0.02, z = 0.90 ± 0.09 e, Vh =+107 ± 3.7 mV (pH 4). The number in parentheses is the number of experiments contributing to each G-V relationship. (C) Voltage-dependent change of BK single channel current induced by extracellular acidification. The filled dots are fractional BK single channel current amplitude determined over the voltage range of ±100 mV when pHO changed from 7 to 4. The Woodhull model (refer to Materials and methods) fit results (solid line) are: kd0 = 0.27 ± 0.02 mM (pH 3.6) and δ = 0.17 e ± 0.02. The prediction of fractional BK single channel current amplitude by the Woodhull model is extended to ±200 mV. Note that the residual single channel current at −120 mV at pHO 4.0 is predicted to be 0.55 of that at pHO 7.0, which is comparable to the Gmax at pHO 4 in panel B. (D) BK G-V curves as those shown in panel B, but with the values at each pHO normalized to the maximum value within that pHO. Boltzmann fit results (solid lines) are Gmax = 0.97 ± 0.01, z = 0.98 ± 0.04 e, Vh = −48.3 ± 1.1 mV (pH 9), Gmax = 0.98 ± 0.01, z = 1.05 ± 0.04 e, Vh = −30.5 ± 1.1 mV (pH 7), Gmax = 0.96 ± 0.01, z = 1.04 ± 0.05 e, Vh = −14.5 ± 1.2 mV (pH 6), Gmax = 0.97 ± 0.01, z = 0.86 ± 0.04 e, Vh =+35.0 ± 1.6 mV (pH 5), Gmax = 1.01 ± 0.02, z = 0.90 ± 0.04 e, Vh =+107.6 ± 1.8 mV (pH 4). The Vh and z values are virtually identical to those determined from the G-Vs in Figure 1B. (E) Dose-response curve of BK gating shift (ΔVh) induced by extracellular H+. Hill equation (ΔVh=ΔVmax/(1+(IC50/[H+])n)) fit result (solid line) is: IC50 = 0.17 ± 0.28 mM (pH 3.8), n = 0.41 ± 0.07.Figure 1—figure supplement 1. BK channel single channel current at various voltages and pHOs.(A–B) Representative current traces for determining BK single channel current amplitude at different conditions. These traces were recorded from different outside-out patches due the large change in PO induced by the change of voltages and pHOs. [Ca2+]in was 300 μM for all these experiments. Closed level is marked by dotted line. All four traces are displayed at same scale for comparison. Scale bar: 20 pA vertical, 20 ms horizontal. (A) Single channel activity of BK channels at −100 mV at pHO 7 (left) or 4 (right). (B) Single channel activity of BK channels at +100 mV at pHO 7 (left) or 4 (right). (C) BK single channel i-V plot at pHO 7 (black dots) and 4 (red dots) over the voltage range of ±100 mV. The number in parentheses is the number of recordings for each data point and the error bars are smaller than the data points. | |
Figure 2. Reduction of BK C-O equilibrium constant L only accounts for a small portion of observed gating shift by extracellular acidification.(A) BK single channel activity recorded from an outside-out patch held at −100 mV perfusing in solutions at pH 7 or pH 4. 10 μM Ca2+ was included in pipette solution. Opening is downward. The nPO of the trace at pH 7 is 0.067. The nPO of the trace at pH 4 is 0.03. Scale bars: 10 pA vertical, 50 ms horizontal. (B) Fractional nPO of BK channels when pHO is changed from 7 to 4 at three negative holding potentials (−100, −120, −140 mV). The number of patches obtained at each potential is listed above each column. There is no significant difference among values determined at these three holding potentials (p>0.05, one-way ANOVA). (C) G-V curves calculated using the H-A model with published parameters: Kd = 11 μM, C = 8, D = 24, E = 2.4, L0 = 10−6, VhC = 156 mV, zj = 0.58 e, zl = 0.3 e (Horrigan and Aldrich, 2002) to highlight the potential impact of changes in L0 (zero voltage C-O equilibrium constant). L0 is scaled down from the published value of 10−6 (black line, L0ctrl) to shift the G-V toward positive potentials. Vh is shifted by +16 mV when L0 is reduced to 0.33 × 10−6 (cyan line, L0ctrl/3). (D) Change of Vh plotted against fold-reduction in L0. The data points are connected by a cubic spline line with no physical meaning. Dotted line marks the change of Vh observed in our experiment when pHO was changed from 7 to 4. | |
Figure 3. Inhibition of BK VSD activation is the primary mechanism for BK gating shifts induced by extracellular H+.(A) BK gating currents recorded from a macropatch perfused at pHO 7 (left) or 4 (right). Test pulses were from −80 to +360 mV in 20 mV increments. Red traces were evoked by 200 mV test pulses. (B) Normalized QC-V relationship of BK at pHO 7 and 4 averaged from five experiments. Gray lines are Boltzmann fits (pH7: zj = 0.46 ± 0.02 e, VhC = 190.1 ± 2.7 mV, pH4: zj = 0.38 ± 0.02 e, VhC = 309.5 ± 2.8 mV). (C) BK G-V relationships at pHO 7 or 4 with 10 μM [Ca2+]in (n = 5). The G-V at pH 7 is fit with H-A model using published parameters (Horrigan and Aldrich, 2002) as initial values. zj and VhC are fixed at the values determined from the QC-V relationship at pH 7. The fit results are: Kd = 3 μM, C = 11, D = 24, E = 1, L0 = 2.7 × 10−6, zl = 0.1 e (black line). The cyan line is a G-V curve calculated using the H-A model with this same set of parameters except that L0 is reduced to 0.9 × 10−6. The red line is a G-V calculated using the H-A model with VhC and zj from the Boltzmann fit of QC-V at pHO 4 and reduced L0 (0.9 × 10−6). | |
Figure 4. BK acidic residues potentially exposed to extracellular medium.(A) mSlo1 homology model based on the cryo-EM structure of liganded Aplysia BK channel (PDB: 5tj6) viewed from the extracellular side. Acidic residues potentially exposed to extracellular solution are rendered as spheres with oxygen and carbon colored in red and gray, respectively. The BK VSDs and PGD are colored in red and blue, respectively. The purple dot in the center is K+. (B) Multiple sequence alignment of transmembrane segments containing extracellularly accessible acidic residues from mouse and Aplysia homologues of Slo1 (mSlo1, aSlo1), Kv channels (Kv1.2, Shaker), and human Nav1.1 and Cav1.1 channels. For the latter two channels, only the DIII VSD segments were included in the alignment. Extracellularly accessible acidic residues conserved among these channels are indicated by boxes. Residues examined in the present study are bold. | |
Figure 5. Acidic residues involved in BK inhibition induced by extracellular H+.(A) Macroscopic currents of mSlo1D153A from an outside-out patch perfused at pHO 7 (left) or pHO 4 (right). The currents of D153A were evoked by steps from −160 to +240 mV with 20 mV increments. All other currents shown in this figure were evoked by steps from −120 to +200 mV (20 mV increments). All pipette (intracellular) solutions contained 300 μM Ca2+. (B) The G-V curves of D153A at pHO 7 or 4. The number of patches contributing to each set of G-V relationship is given in parentheses in this and following G-V plots. Boltzmann fit results (grey lines) are z = 0.70 ± 0.03 e, Vh = 179.4 ± 2.4 mV (pH 7), z = 0.78 ± 0.03 e, Vh = 186.4 ± 2.2 mV (pH 4), Gmax = 1.16 ± 0.03. Dotted lines in this and following G-V plots are the G-V curves of WT channels at pH7 (black) or 4 (red) with 300 μM [Ca2+]in (C) Macroscopic currents of mSlo1D133A at pHO 7 (left) or pH 4 (right). (D) The G-V curves of D133A at pHO 7 or 4. Boltzmann fit results (grey lines) are z = 0.76 ± 0.03 e, Vh = 66.1 ± 1.7 mV (pH 7), z = 0.81 ± 0.03 e, Vh = 116.2 ± 1.6 mV (pH 4), Gmax = 1.00 ± 0.02. (E) Macroscopic currents of mSlo1D147A at pHO 7 (left) or pH 4 (right). (F) The G-V curves of D147A at pHO 7 or 4. Boltzmann fit results (grey lines) are z = 0.70 ± 0.04 e, Vh = 60.8 ± 2.6 mV (pH 7), z = 0.77 ± 0.05 e, Vh = 126.7 ± 2.3 mV (pH 4), Gmax = 0.99 ± 0.02. (G) Macroscopic currents of mSlo1D133A147A at pHO 7 (left) or pH 4 (right). (H) The G-V curves of mSlo1D133AD147A at pHO 7 or 4. Boltzmann fit results (grey lines) are z = 0.74 ± 0.04 e, Vh = 115.6 ± 2.3 mV (pH 7), z = 0.78 ± 0.04 e, Vh = 138.9 ± 2.1 mV (pH 4), Gmax = 1.07 ± 0.03. (I) Change of gating equilibrium free energy by extracellular acidification(ΔG0pH) calculated from the Boltzmann fits of G-V curves. The number of experiments for each construct is listed above each column. (J) The change of gating equilibrium free energy by alanine substitution (ΔG0MUT) plotted against the change of gating equilibrium free energy by extracellular acidification (ΔG0pH). The solid line is a linear fit with R of 0.96. Standard error in ΔG0MUT (αΔG0MUT) is calculated according to: αΔG0MUT = (α2G0WT - α 2G0MUT) 1/2. | |
Figure 6. Potential interactions involving the three key residues in the BK VSD.(A) The VSDs of metal-free (red) and liganded (blue) mSlo1 structures with three key acidic residues rendered as ball-and-chain and three conserved S4 arginines (homologous to Shaker R2, R3 and R4) rendered as sticks. The four dark purple dots are K+s in the BK selectivity filter, which are included as a reference for vertical displacement. The two structures are superimposed by the pore-loop. (B) D153 may form a hydrogen bond with R207 in the metal-free mSlo1 structure. The distance between the side chains of D153 and R210 is more than 6 Å, which is too far for hydrogen bonding. (C) D153 may form a hydrogen bond with R210 in the liganded mSlo1 structure. The distance between the side chains of D153 and R207 is more than 4 Å, which is also beyond the limit of effective hydrogen bonding. (D–E) The distances between the side chains of D133 and R207 in both the metal-free (D) and the liganded (E) structures are within 3 Å, allowing potential interaction between D133 and R207 in both conformations. |
References [+] :
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
Bailey, Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet. 2006, Pubmed
Brenner, Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. 2000, Pubmed
Brenner, BK channel beta4 subunit reduces dentate gyrus excitability and protects against temporal lobe seizures. 2005, Pubmed
Bright, Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity. 2016, Pubmed
Cao, BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release. 2015, Pubmed
Carrasquel-Ursulaez, Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel. 2015, Pubmed , Xenbase
Christensen, Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential. 1987, Pubmed
Claydon, A direct demonstration of closed-state inactivation of K+ channels at low pH. 2007, Pubmed , Xenbase
Claydon, Inhibition of the K+ channel kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation. 2000, Pubmed , Xenbase
Claydon, Closed-state inactivation induced in K(V)1 channels by extracellular acidification. 2008, Pubmed
Cook, Lowering of pHi inhibits Ca2+-activated K+ channels in pancreatic B-cells. , Pubmed
Deutsch, Modulation of K+ currents in human lymphocytes by pH. 1989, Pubmed
DiChiara, Distinct effects of Ca2+ and voltage on the activation and deactivation of cloned Ca(2+)-activated K+ channels. 1995, Pubmed , Xenbase
FRANKENHAEUSER, The action of calcium on the electrical properties of squid axons. 1957, Pubmed
Forsyth, Empirical relationships between protein structure and carboxyl pKa values in proteins. 2002, Pubmed
Gilly, Divalent cations and the activation kinetics of potassium channels in squid giant axons. 1982, Pubmed
Grimm, Identification and localization of BK-beta subunits in the distal nephron of the mouse kidney. 2007, Pubmed
Guarina, Low pHo boosts burst firing and catecholamine release by blocking TASK-1 and BK channels while preserving Cav1 channels in mouse chromaffin cells. 2017, Pubmed
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, 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
Haug, Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part II: Neutralization of aspartate 292 reduces long channel openings and gating current slow component. 2004, Pubmed , Xenbase
Heitzmann, No potassium, no acid: K+ channels and gastric acid secretion. 2007, Pubmed
Hille, Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH. 1975, Pubmed
Hite, Structural basis for gating the high-conductance Ca2+-activated K+ channel. 2017, 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
Horrigan, Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+). 1999, Pubmed
Horrigan, Mg2+ enhances voltage sensor/gate coupling in BK channels. 2008, Pubmed , Xenbase
Hoshi, Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca²⁺-dependent K⁺ channels. 2013, Pubmed
Hoshi, Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA. 2013, Pubmed
Hoshi, Initial steps in the opening of a Shaker potassium channel. 2012, Pubmed
Hou, Modulation of BKCa channel gating by endogenous signaling molecules. 2009, Pubmed
Hou, Reciprocal regulation of the Ca2+ and H+ sensitivity in the SLO1 BK channel conferred by the RCK1 domain. 2008, Pubmed
Hou, The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels. 2008, 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
Johnson, The position of lysosomes within the cell determines their luminal pH. 2016, Pubmed
Kwan, Single channel analysis reveals different modes of Kv1.5 gating behavior regulated by changes of external pH. 2006, Pubmed
Latorre, Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. 1982, Pubmed
Li, Nuclear BK channels regulate gene expression via the control of nuclear calcium signaling. 2014, Pubmed
Long, Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. 2007, Pubmed
Ma, Role of charged residues in the S1-S4 voltage sensor of BK channels. 2006, Pubmed , Xenbase
Ma, An extracellular Cu2+ binding site in the voltage sensor of BK and Shaker potassium channels. 2008, Pubmed , Xenbase
Magleby, Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle. 1983, Pubmed
Meredith, BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output. 2006, Pubmed
Mindell, Lysosomal acidification mechanisms. 2012, Pubmed
Pathak, Closing in on the resting state of the Shaker K(+) channel. 2007, Pubmed
Pettersen, UCSF Chimera--a visualization system for exploratory research and analysis. 2004, Pubmed
Prole, Mechanisms underlying modulation of neuronal KCNQ2/KCNQ3 potassium channels by extracellular protons. 2003, Pubmed
Reinhart, Modulation of calcium-activated potassium channels from rat brain by protein kinase A and phosphatase 2A. 1991, Pubmed
Robitaille, Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. 1993, Pubmed
Rosenblatt, Distribution of Ca2+-activated K+ channel isoforms along the tonotopic gradient of the chicken's cochlea. 1997, Pubmed
Sali, Comparative protein modelling by satisfaction of spatial restraints. 1993, Pubmed
Sandle, Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis. 2007, Pubmed
Schreiber, A novel calcium-sensing domain in the BK channel. 1997, Pubmed , Xenbase
Schubert, Protons inhibit the BK(Ca) channel of rat small artery smooth muscle cells. 2001, Pubmed
Shi, Mechanism of magnesium activation of calcium-activated potassium channels. 2002, Pubmed
Shi, Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels. 2001, Pubmed , Xenbase
Singh, MitoBK(Ca) is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location. 2013, Pubmed
Sørensen, The secretory KCa1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence. 2011, Pubmed
Tanaka, Beta1-subunit of MaxiK channel in smooth muscle: a key molecule which tunes muscle mechanical activity. 2004, Pubmed
Tang, Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels. 2003, Pubmed
Tao, A gating charge transfer center in voltage sensors. 2010, Pubmed , Xenbase
Tao, Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. 2017, Pubmed
Tian, Distinct stoichiometry of BKCa channel tetramer phosphorylation specifies channel activation and inhibition by cAMP-dependent protein kinase. 2004, Pubmed
Tian, Reversible tyrosine protein phosphorylation regulates large conductance voltage- and calcium-activated potassium channels via cortactin. 2008, Pubmed
Tiwari-Woodruff, Voltage-dependent structural interactions in the Shaker K(+) channel. 2000, Pubmed , Xenbase
Trapani, Effect of external pH on activation of the Kv1.5 potassium channel. 2003, Pubmed
Tseng-Crank, Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain. 1994, Pubmed , Xenbase
Wagner, Renal vacuolar H+-ATPase. 2004, 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
Wang, A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores. 2017, Pubmed
Wellman, Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels. 2003, Pubmed
Woodhull, Ionic blockage of sodium channels in nerve. 1973, 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, 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
Xu, Lysosomal physiology. 2015, Pubmed
Yan, LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium. 2010, Pubmed
Yifrach, Energetics of pore opening in a voltage-gated K(+) channel. 2002, Pubmed , Xenbase
Zhang, Modulation of Kv1.5 potassium channel gating by extracellular zinc. 2001, Pubmed
Zhang, Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition. 2003, 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
Zhou, Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism. 2014, Pubmed , Xenbase
Zhou, Glycine311, a determinant of paxilline block in BK channels: a novel bend in the BK S6 helix. 2010, Pubmed , Xenbase