scholarly journals Voltage and Ca2+ Activation of Single Large-Conductance Ca2+-Activated K+ Channels Described by a Two-Tiered Allosteric Gating Mechanism

2000 ◽  
Vol 116 (1) ◽  
pp. 75-100 ◽  
Author(s):  
Brad S. Rothberg ◽  
Karl L. Magleby
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Mechanism ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
The Mean

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, qeff, of 2.3 ± 0.6 eo). Estimates of qeff were little changed for intracellular Ca2+ (Ca2+i) ranging from 0.0003 to 1,024 μM. Increasing Ca2+i from 0.03 to 1,024 μM shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2+i for Ca2+i ≤ 0.03 μM, indicating that the channel can be activated in the absence of Ca2+i. Open and closed dwell-time distributions for data obtained at different Ca2+i and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (qeff = −0.5 eo) and an increase in the mean opening rate (qeff = 1.8 eo), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+i (∼0 through 1,024 μM), voltage (+80 to −80 mV), and Po (10−4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.

scholarly journals The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

1999 ◽  
Vol 113 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Α Subunit ◽  
Closed Intervals ◽  
Single Channel Analysis ◽  
Channel Analysis ◽  
The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

scholarly journals Anomalous L-Type Calcium Channels of Rat Spinal Motoneurons

1999 ◽  
Vol 113 (5) ◽  
pp. 679-694 ◽  
Author(s):  
Bruno Hivert ◽  
Siro Luvisetto ◽  
Anacleto Navangione ◽  
Angelita Tottene ◽  
Daniela Pietrobon
Keyword(s):  
Calcium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Spinal Motoneurons ◽  
Relative Probability ◽  
Open Probability ◽  
Voltage Dependent ◽  
The Mean ◽  
Cytosolic Factor ◽  
Rule Out

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range −60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.

scholarly journals Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels

1999 ◽  
Vol 114 (2) ◽  
pp. 215-242 ◽  
Author(s):  
Max Kanevsky ◽  
Richard W. Aldrich
Keyword(s):  
Voltage Dependence ◽  
Gating Current ◽  
Wild Type ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Shaker Potassium Channels

The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh5, differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67–73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399–4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh5 revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799–1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh5 mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

scholarly journals Gating Kinetics of Single Large-Conductance Ca2+-Activated K+ Channels in High Ca2+ Suggest a Two-Tiered Allosteric Gating Mechanism✪

1999 ◽  
Vol 114 (1) ◽  
pp. 93-124 ◽  
Author(s):  
Brad S. Rothberg ◽  
Karl L. Magleby
Keyword(s):  
Dwell Time ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Kinetics ◽  
Open Probability ◽  
Kinetic Schemes ◽  
Gating Mechanism ◽  
Open States

The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca2+i ∼ 100 μM. Increasing Ca2+i further to 1,024 μM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 μM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

2007 ◽  
Vol 293 (1) ◽  
pp. F236-F244 ◽  
Author(s):  
Ling Yu ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Heavy Metal ◽  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Histidine Residues ◽  
Voltage Dependent ◽  
Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

scholarly journals Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 309-328 ◽  
Author(s):  
Zhongming Ma ◽  
Xing Jian Lou ◽  
Frank T. Horrigan
Keyword(s):  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Voltage Sensing ◽  
Kv Channels ◽  
Gating Charge ◽  
Charged Residues ◽  
Sensor Activation

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

scholarly journals The BK channel gating ring is strongly coupled to the voltage sensor

2018 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Divalent Ions ◽  
Gating Currents ◽  
Open Probability ◽  
Voltage Dependent ◽  
Tightly Coupled

ABSTRACTThe open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

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