scholarly journals Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel.

1996 ◽  
Vol 108 (4) ◽  
pp. 237-250 ◽  
Author(s):  
T Y Chen ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
Voltage Dependence ◽  
High Rate ◽  
Charge Movement ◽  
Ion Conduction ◽  
Closed State ◽  
Cl Channel ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Fast Gating

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)-liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.

scholarly journals Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

1988 ◽  
Vol 91 (3) ◽  
pp. 335-349 ◽  
Author(s):  
R MacKinnon ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
External Solution ◽  
Dissociation Rate ◽  
K Channel ◽  
Internal Solution ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
K Concentration ◽  
A Site ◽  
Low K

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

scholarly journals The Role of a Conserved Lysine in Chloride- and Voltage-dependent ClC-0 Fast Gating

2007 ◽  
Vol 130 (4) ◽  
pp. 351-363 ◽  
Author(s):  
Anita M. Engh ◽  
José D. Faraldo-Gómez ◽  
Merritt Maduke
Keyword(s):  
Structural Changes ◽  
Computational Analysis ◽  
Voltage Dependence ◽  
Structural Information ◽  
Chloride Binding ◽  
Gate Opening ◽  
Voltage Dependent ◽  
Fast Gating ◽  
Homology Models

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.

scholarly journals Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.

1987 ◽  
Vol 90 (3) ◽  
pp. 427-449 ◽  
Author(s):  
C Miller ◽  
R Latorre ◽  
I Reisin
Keyword(s):  
Lipid Bilayers ◽  
Dissociation Rate ◽  
K Channel ◽  
Single Channels ◽  
Closed Channel ◽  
Association Rate ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
High Conductance

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.

scholarly journals Modulation of the Voltage Sensor of L-type Ca2+ Channels by Intracellular Ca2+

2004 ◽  
Vol 123 (5) ◽  
pp. 555-571 ◽  
Author(s):  
Dmytro Isaev ◽  
Karisa Solt ◽  
Oksana Gurtovaya ◽  
John P. Reeves ◽  
Roman Shirokov
Keyword(s):  
Intracellular Calcium ◽  
Calcium Binding ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Wild Type ◽  
Calcium Binding Site ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Charge Movements

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (≥100 μM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from ∼0.1 to 100–300 μM sped up the conversion of the gating charge into the negatively distributed mode 10–100-fold. Since the “IQ-AA” mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the “IQ” motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Δ1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.

scholarly journals Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

2005 ◽  
Vol 126 (5) ◽  
pp. 419-428 ◽  
Author(s):  
Donato del Camino ◽  
Max Kanevsky ◽  
Gary Yellen
Keyword(s):  
Conformational Changes ◽  
Charge Movement ◽  
Ion Flow ◽  
K Channels ◽  
Closed State ◽  
Channel Opening ◽  
Activated State ◽  
Form Part ◽  
Voltage Dependent ◽  
Flow Through

Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

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 Broadband Tuning the Voltage Dependence of a Sodium Channel

2020 ◽  
Author(s):  
Eedann McCord ◽  
Goragot Wisedchaisri ◽  
William A. Catterall
Keyword(s):  
Amino Acid ◽  
Sodium Channel ◽  
Voltage Dependence ◽  
Membrane Potentials ◽  
Channel Structure ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Amino Acid Residues ◽  
Gating Currents ◽  
Voltage Dependent

ABSTRACTVoltage-gated sodium channels initiate action potentials in prokaryotes and in many eukaryotic cells, including vertebrate nerve and muscle. Their activation is steeply voltage-dependent, but it is unclear how the voltage sensitivity is set or whether it can be broadly shifted to positive voltages. Here we show that the voltage dependence of activation (VA) of the ancestral bacterial sodium channel NaVAb can be progressively shifted from −118 mV to +35 mV in chimeras with increasing numbers of amino acid residues from the extracellular half of the voltage sensor of human NaV1.7 channels. In a minimal chimera in which only 32 residues were transferred, we analyzed the effects of six additional mutations of conserved amino acid residues singly, in pairs, and as triple mutations. The resulting chimeric mutants exhibited a broad range of voltage sensitivity from VA=−118 mV to VA=+120 mV. Three mutations (N48K, L112A, and M119V) shifted VA to +61 mV when substituted in NaVAb itself, and substitution of two additional Cys residues in the Cys-free background of NaVAb further shifted VA to +105 mV. In these mutants, measurement of gating currents revealed that the voltage dependence of gating charge movement (VQ) shifted to positive membrane potentials as much or more than VA, confirming that the gating charges are trapped in their resting positions by these VA-shifting mutations. Our results demonstrate broadband shifting of VA and VQ of a sodium channel across a range of 240 mV and provide a toolbox of methods and constructs to analyze sodium channel structure and function in the resting state at 0 mV and in activated states at positive membrane potentials.GRAPHICAL ABSTRACTThe complete range of broadband tuning of voltage-dependent activation of a sodium channel.

scholarly journals Ion Transport Through Excitability-Inducing Material (EIM) Channels in Lipid Bilayer Membranes

1972 ◽  
Vol 60 (1) ◽  
pp. 72-85 ◽  
Author(s):  
Ramon Latorre ◽  
Gerald Ehrenstein ◽  
Harold Lecar
Keyword(s):  
Aqueous Solution ◽  
Ion Transport ◽  
Lipid Bilayers ◽  
Voltage Dependence ◽  
The Other ◽  
Lipid Bilayer Membranes ◽  
Relative Permeabilities ◽  
Bilayer Membranes ◽  
Voltage Dependent ◽  
Ionic Mobilities

Two different methods were used to determine the relative permeability and the voltage-dependent conductance of several different cations in excitability-inducing material (EIM)-doped lipid bilayers. In one method, the conductances of individual channels were measured for Li, Na, K, Cs, NH4, and Ca, and in the other method biionic potentials of a membrane with many channels were measured for Li, Na, K, Cs, and Rb. The experimental results for the two methods are in agreement. The relative permeabilities are proportional to the ionic mobilities in free aqueous solution. The voltage dependence of the conductance is the same for all cations measured.

scholarly journals Separation of intramembrane charging components in low-calcium solutions in frog skeletal muscle.

1991 ◽  
Vol 98 (2) ◽  
pp. 249-263 ◽  
Author(s):  
C L Huang
Keyword(s):  
Voltage Dependence ◽  
Charge Movement ◽  
Pharmacological Effects ◽  
Frog Skeletal Muscle ◽  
Calcium Signals ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Charge Movements

The inactivation of charge movement components by small (-100 to -70 mV) shifts in holding potential was examined in voltage-clamped intact amphibian muscle fibers in low [Ca2+], Mg(2+)-containing solutions. The pulse protocols used both large voltage excursions and smaller potential steps that elicited prolonged (q gamma) transients. Charge species were distinguished through the pharmacological effects of tetracaine. These procedures confirmed earlier observations in cut fibers and identified the following new properties of the q gamma charge. First, q gamma, previously defined as the tetracaine-sensitive charge, is also the component primarily responsible for the voltage-dependent inactivation induced by conditions of low extracellular [Ca2+]. Second, this inactivation separates a transient that includes a "hump" component and which has kinetics and a voltage dependence distinct from the monotonic decay that remains. Third, q gamma, previously associated with delayed charge movements, can also contribute significant charge transfer at early times. These findings suggest that the parallel inhibition of calcium signals and charge movements reported in low [Ca2+] solutions arises from influences on q gamma charge (Brum et al., 1988a, b). They also reconcile reports that implicate tetracaine-sensitive (q gamma) charge in excitation-contraction coupling with evidence that early intramembrane events are also involved in this process (Pizarro et al., 1989). Finally, they are relevant to hypotheses of possible feedback or feed-forward roles of q gamma in excitation-contraction coupling.

scholarly journals Voltage-dependent block of charge movement components by nifedipine in frog skeletal muscle.

1990 ◽  
Vol 96 (3) ◽  
pp. 535-557 ◽  
Author(s):  
C L Huang
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Voltage Dependence ◽  
Membrane Capacitance ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Membrane Charge ◽  
Capacitance Voltage ◽  
Voltage Dependent ◽  
Dependent Inhibition

Potential-dependent inhibition of charge movement components by nifedipine was studied in intact, voltage-clamped, frog skeletal muscle fibers. Available charge was reduced by small shifts in holding potential (from -100 mV to -70 mV) in 2 microM nifedipine, without changes in the capacitance deduced from control (-120 mV to -100 mV) voltage steps made at a fully polarized (-100 mV) holding potential. These voltage-dependent effects did not occur in lower (0-0.5 microM) nifedipine concentrations. The voltage dependence of membrane capacitance at higher (10 microM) nifedipine concentrations was reduced even in fully polarized fibers, but shifting the holding voltage produced no further block. Voltage-dependent inhibition by nifedipine was associated with a fall in available charge, and a reduction in the charge and capacitance-voltage relationships and of late (q gamma) charging transients. It thus separated a membrane-capacitance with a distinct and steep steady-state voltage dependence. Tetracaine (2 mM) reduced voltage-dependent membrane capacitance and nonlinear charge more than did nifedipine. However, nifedipine did not exert voltage-dependent effects on charging currents, membrane capacitance, or inactivation of tetracaine-resistant (q beta) charge. This excludes participation of q beta, or the membrane charge as a whole, from the voltage-dependent effects of nifedipine. Rather, the findings suggest that the charge susceptible to potential-dependent block by nifedipine falls within the tetracaine-sensitive (q gamma) category of intramembrane charge.

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