scholarly journals Mechanism of the Inhibition of Ca2+-Activated Cl− Currents by Phosphorylation in Pulmonary Arterial Smooth Muscle Cells

2006 ◽  
Vol 128 (1) ◽  
pp. 73-87 ◽  
Author(s):  
Jeff E. Angermann ◽  
Amy R. Sanguinetti ◽  
James L. Kenyon ◽  
Normand Leblanc ◽  
Iain A. Greenwood
Keyword(s):  
Chloride Channels ◽  
Voltage Dependence ◽  
Critical Voltage ◽  
Kinetic Properties ◽  
Calcium Dependent ◽  
Membrane Rupture ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pulmonary Arterial ◽  
The Impact

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.

scholarly journals Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence.

1994 ◽  
Vol 104 (2) ◽  
pp. 311-336 ◽  
Author(s):  
D H Cox ◽  
K Dunlap
Keyword(s):  
Voltage Dependence ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Drg Neurons ◽  
Rapid Phase ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Dependent Processes

We have studied the inactivation of high-voltage-activated (HVA), omega-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage-dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes

1987 ◽  
Vol 231 (1262) ◽  
pp. 27-36 ◽  
Keyword(s):  
Chloride Channels ◽  
Calcium Influx ◽  
Inositol Trisphosphate ◽  
Ringer Solution ◽  
Chloride Ions ◽  
Bathing Solution ◽  
Calcium Dependent ◽  
Large Numbers ◽  
Voltage Dependent ◽  
Oocyte Membrane

Injection of inositol trisphosphate (IP 3 ) into oocytes of Xenopus laevis induces the appearance of a transient inward ( T in ) current on hyper­polarization of the membrane. This current is carried largely by chloride ions, but is shown to depend on extracellular calcium, because it is abolished by removal of calcium in the bathing fluid or by addition of manganese. Recordings with aequorin as an intracellular calcium indi­cator show that a calcium influx is activated by hyperpolarization after intracellular injection of IP 3 as well as after activation of neurotrans­mitter receptors thought to mediate a rise in IP 3 . Furthermore, by substituting barium for calcium in the bathing solution, inward barium currents can be recorded during hyperpolarization. We conclude that intracellular IP 3 modulates the activity of a class of calcium channels, so as to allow an influx of calcium on hyperpolarization. In normal Ringer solution this then leads to the generation of a chloride current, because of the large numbers of calcium-dependent chloride channels in the oocyte membrane.

scholarly journals Heme Regulates Allosteric Activation of the Slo1 BK Channel

2005 ◽  
Vol 126 (1) ◽  
pp. 7-21 ◽  
Author(s):  
Frank T. Horrigan ◽  
Stefan H. Heinemann ◽  
Toshinori Hoshi
Keyword(s):  
Divalent Cations ◽  
Ionic Currents ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Gating Currents ◽  
Calcium Dependent ◽  
Channel Opening ◽  
Activation Gate ◽  
Voltage Dependent

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state.

scholarly journals N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle.

1985 ◽  
Vol 86 (5) ◽  
pp. 601-611 ◽  
Author(s):  
B S Pallotta
Keyword(s):  
Skeletal Muscle ◽  
Potassium Channels ◽  
Voltage Dependence ◽  
Calcium Sensitivity ◽  
Rat Skeletal Muscle ◽  
Open Probability ◽  
Calcium Dependent ◽  
Channel Opening ◽  
Dependent Component ◽  
Calcium Activated Potassium Channels

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.

scholarly journals Fine Gating Properties of Channels Responsible for Persistent Sodium Current Generation in Entorhinal Cortex Neurons

2002 ◽  
Vol 120 (6) ◽  
pp. 855-873 ◽  
Author(s):  
Jacopo Magistretti ◽  
Angel Alonso
Keyword(s):  
Entorhinal Cortex ◽  
Voltage Dependence ◽  
Burst Duration ◽  
The Other ◽  
Na Channel ◽  
Kinetic Properties ◽  
Channel Activity ◽  
Other Hand ◽  
Voltage Dependent ◽  
The Mean

The gating properties of channels responsible for the generation of persistent Na+ current (INaP) in entorhinal cortex layer II principal neurons were investigated by performing cell-attached, patch-clamp experiments in acutely isolated cells. Voltage-gated Na+-channel activity was routinely elicited by applying 500-ms depolarizing test pulses positive to −60 mV from a holding potential of −100 mV. The channel activity underlying INaP consisted of prolonged and frequently delayed bursts during which repetitive openings were separated by short closings. The mean duration of openings within bursts was strongly voltage dependent, and increased by e times per every ∼12 mV of depolarization. On the other hand, intraburst closed times showed no major voltage dependence. The mean duration of burst events was also relatively voltage insensitive. The analysis of burst-duration frequency distribution returned two major, relatively voltage-independent time constants of ∼28 and ∼190 ms. The probability of burst openings to occur also appeared largely voltage independent. Because of the above “persistent” Na+-channel properties, the voltage dependence of the conductance underlying whole-cell INaP turned out to be largely the consequence of the pronounced voltage dependence of intraburst open times. On the other hand, some kinetic properties of the macroscopic INaP, and in particular the fast and intermediate INaP-decay components observed during step depolarizations, were found to largely reflect mean burst duration of the underlying channel openings. A further INaP decay process, namely slow inactivation, was paralleled instead by a progressive increase of interburst closed times during the application of long-lasting (i.e., 20 s) depolarizing pulses. In addition, long-lasting depolarizations also promoted a channel gating modality characterized by shorter burst durations than normally seen using 500-ms test pulses, with a predominant burst-duration time constant of ∼5–6 ms. The above data, therefore, provide a detailed picture of the single-channel bases of INaP voltage-dependent and kinetic properties in entorhinal cortex layer II neurons.

scholarly journals Mutations in the S4 Region Isolate the Final Voltage-dependent Cooperative Step in Potassium Channel Activation

1999 ◽  
Vol 113 (3) ◽  
pp. 389-414 ◽  
Author(s):  
Jennifer L. Ledwell ◽  
Richard W. Aldrich
Keyword(s):  
Voltage Dependence ◽  
Ionic Current ◽  
Amino Acid Sequences ◽  
Total Charge ◽  
Charge Movement ◽  
Activation Pathway ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Rate Limiting ◽  
Cooperative Transition

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% (∼1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.

scholarly journals Coupling of Voltage Sensing to Channel Opening Reflects Intrasubunit Interactions in Kv Channels

2004 ◽  
Vol 125 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Alain J. Labro ◽  
Adam L. Raes ◽  
Dirk J. Snyders
Keyword(s):  
Voltage Dependence ◽  
Voltage Sensing ◽  
Α Subunit ◽  
K Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Subunit Stoichiometry ◽  
Voltage Dependent ◽  
Tetrameric Structure

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.

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 Permeation and Block of the Skeletal Muscle Chloride Channel, ClC-1, by Foreign Anions

1998 ◽  
Vol 111 (5) ◽  
pp. 653-665 ◽  
Author(s):  
G.Y. Rychkov ◽  
M. Pusch ◽  
M.L. Roberts ◽  
T.J. Jentsch ◽  
A.H. Bretag
Keyword(s):  
Skeletal Muscle ◽  
Binding Site ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Single Channel ◽  
Hydrophobic Interactions ◽  
Channel Pore ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Significant Block

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl− as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3−, BrO3−); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl− at the regulatory binding site but impair Cl− passage through the channel pore (Br−, NO3−, ClO3−, I−, ClO4−, SCN−). The permeability sequence for rClC-1, SCN− ∼ ClO4− > Cl− > Br− > NO3− ∼ ClO3− > I− >> BrO3− > HCO3− >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN− ∼ ClO4− > I− > NO3− ∼ ClO3− ∼ methanesulfonate > Br− > cyclamate > BrO3− > HCO3− > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl− and SCN− or ClO4− suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.

scholarly journals Allosteric Gating of a Large Conductance Ca-activated K+ Channel

1997 ◽  
Vol 110 (3) ◽  
pp. 257-281 ◽  
Author(s):  
D.H. Cox ◽  
J. Cui ◽  
R.W. Aldrich
Keyword(s):  
Steady State ◽  
General Scheme ◽  
Kinetic Scheme ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Kinetic Properties ◽  
Allosteric Proteins ◽  
Channel Opening ◽  
Voltage Dependent

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.

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