scholarly journals Activation of Shaker Potassium Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 313-342 ◽  
Author(s):  
N.E. Schoppa ◽  
F.J. Sigworth
Keyword(s):  
Single Channel ◽  
Conformational Stability ◽  
Ionic Currents ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Shaker Potassium Channel ◽  
Channel Opening ◽  
Tetrameric Structure ◽  
Charge Movements

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker's symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e0), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e0). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker's activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.

scholarly journals Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

1996 ◽  
Vol 107 (4) ◽  
pp. 515-534 ◽  
Author(s):  
C L Huang
Keyword(s):  
Steady State ◽  
Time Course ◽  
Striated Muscle ◽  
Ionic Currents ◽  
Time Dependent ◽  
Total Charge ◽  
Charge Movement ◽  
Charge Voltage ◽  
Charge Movements ◽  
State Charge

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembrane charge were investigated in intact voltage-clamped frog skeletal muscle fibers under conditions that minimized time-dependent ionic currents. A hypothesis that RyR gating is allosterically coupled to configurational changes in dihydropyridine receptors (DHPRs) would predict that such interactions are reciprocal and that RyR modification should influence intramembrane charge. Both agents indeed modified the time course of charging transients at 100-200-microM concentrations. They independently abolished the delayed charging phases shown by q gamma currents, even in fibers held at fully polarized, -90-mV holding potentials; such waveforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-dependent currents. The steady-state charge transfers nevertheless remained equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(VT), shifted by approximately +10 mV, particularly through those test potentials at which delayed q gamma currents normally took place but retained steepness factors (k approximately 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q gamma contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Qmax (VH), which also retained similarly high voltage sensitivities (k approximately 7.0 to 9.0 mV). RyR antagonists also preserved the separate identities of q gamma and q beta species, whether defined by their steady-state voltage dependence or inactivation or pharmacological properties. Thus, tetracaine (2 mM) reduced the available steady-state charge movement and gave shallow Q(VT) (k approximately 14 to 16 mV) and Qmax (VH) (k approximately 14 to 17 mV) curves characteristic of q beta charge. These features persisted with exposure to test agent. Finally, q gamma charge movements showed steep voltage dependences with both activation (k approximately 4.0 to 6.5 mV) and inactivation characteristics (k approximately 4.3 to 6.6 mV) distinct from those shown by the remaining q beta charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters q gamma kinetics while preserving the separate identities of steady-state q beta and q gamma charge. These findings permit a mechanism by which transverse tubular voltage provides the primary driving force for configurational changes in DHPRs, which might produce q gamma charge movement. However, they attribute its kinetic complexities to the reciprocal allosteric coupling by which DHPR voltage sensors and RyR-Ca2+ release channels might interact even though these receptors reside in electrically distinct membranes. RyR modification then would still permit tubular voltage change to drive net q gamma charge transfer but would transform its complex waveforms into simple exponential decays.

scholarly journals Sodium channel gating currents in frog skeletal muscle.

1983 ◽  
Vol 82 (5) ◽  
pp. 679-701 ◽  
Author(s):  
D T Campbell
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Time Course ◽  
Channel Gating ◽  
Total Charge ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Gating Currents ◽  
Gating Mechanism ◽  
Charge Movements

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.

scholarly journals Shaker potassium channel gating. III: Evaluation of kinetic models for activation.

1994 ◽  
Vol 103 (2) ◽  
pp. 321-362 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
R W Aldrich
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Ionic Currents ◽  
Single Step ◽  
Gating Currents ◽  
Open Probability ◽  
Shaker Potassium Channel ◽  
Single Channel Currents

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

scholarly journals Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature.

1991 ◽  
Vol 98 (2) ◽  
pp. 287-314 ◽  
Author(s):  
C S Hui
Keyword(s):  
Sarcomere Length ◽  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Total Charge ◽  
Charge Movement ◽  
Time To Peak ◽  
Gap Technique ◽  
Different Temperatures ◽  
Charge Movements ◽  
Kinetics Of

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.

scholarly journals Cooh-Terminal Truncated Alpha1S Subunits Conduct Current Better than Full-Length Dihydropyridine Receptors

2000 ◽  
Vol 116 (3) ◽  
pp. 341-348 ◽  
Author(s):  
James A. Morrill ◽  
Stephen C. Cannon
Keyword(s):  
Skeletal Muscle ◽  
Expression System ◽  
Ionic Currents ◽  
Full Length ◽  
Charge Movement ◽  
Functional Roles ◽  
Dihydropyridine Receptors ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Gated Channels

Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca2+ channels and as voltage sensors for coupling membrane depolarization to release of Ca2+ from the sarcoplasmic reticulum. In skeletal muscle, the principal or α1S subunit occurs in full-length (∼10% of total) and post-transcriptionally truncated (∼90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized α1S subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (α1S) or truncated (α1SΔC) α subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Qmax 80–150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in α1S, unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba2+ currents were small relative to Qmax. The truncated α1SΔC protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the α1S subunit conduct the majority of the L-type Ca2+ current in skeletal muscle. Our data also suggest that the carboxyl terminus of the α1S subunit modulates the coupling between charge movement and channel opening.

Observations on intramembrane charge movements in skeletal muscle

1975 ◽  
Vol 270 (908) ◽  
pp. 507-513 ◽  
Keyword(s):  
Ionic Currents ◽  
Charge Movement ◽  
Electrical Excitation ◽  
Signal Averaging ◽  
Gating Current ◽  
Residual Currents ◽  
Contraction Coupling ◽  
Intramembrane Charge Movement ◽  
Charge Movements ◽  
Averaging Techniques

Using signal-averaging techniques, one can record small membrane currents which remain even after blockage of the ionic currents which accompany electrical excitation in muscle. These residual currents probably represent the reorientation of charged molecules inside the membrane in response to a change in membrane potential. Two operationally separable types of intramembrane charge movement in muscle are described, one of which may play a role in excitation—contraction coupling. Studies of tetrodotoxin binding to muscle indicate that ‘sodium gating current’ is unlikely to contribute significantly to either type of charge movement.

scholarly journals On the Mechanism by which 4-Aminopyridine Occludes Quinidine Block of the Cardiac K+ Channel, hKv1.5

1998 ◽  
Vol 111 (4) ◽  
pp. 539-554 ◽  
Author(s):  
Fred S.P. Chen ◽  
David Fedida
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
K Channel ◽  
Gating Currents ◽  
Channel Activation ◽  
Gating Charge ◽  
Channel Opening ◽  
Conformational Rearrangements ◽  
A Site

4-Aminopyridine (4-AP) binds to potassium channels at a site or sites in the inner mouth of the pore and is thought to prevent channel opening. The return of hKv1.5 off-gating charge upon repolarization is accelerated by 4-AP and it has been suggested that 4-AP blocks slow conformational rearrangements during late closed states that are necessary for channel opening. On the other hand, quinidine, an open channel blocker, slows the return or immobilizes off-gating charge only at opening potentials (>−25 mV). The aim of this study was to use quini-dine as a probe of open channels to test the kinetic state of 4-AP-blocked channels. In the presence of 0.2–1 mM 4-AP, quinidine slowed charge return and caused partial charge immobilization, corresponding to an increase in the Kd of ∼20-fold. Peak off-gating currents were reduced and decay was slowed ∼2- to 2.5-fold at potentials negative to the threshold of channel activation and during depolarizations shorter than normally required for channel activation. This demonstrated access of quinidine to 4-AP-blocked channels, a lack of competition between the two drugs, and implied allosteric modulation of the quinidine binding site by 4-AP resident within the channel. Single channel recordings also showed that quinidine could modulate the 4-AP-induced closure of the channels, with the result that frequent channel reopenings were observed when both drugs were present. We propose that 4-AP-blocked channels exist in a partially open, nonconducting state that allows access to quinidine, even at more negative potentials and during shorter depolarizations than those required for channel activation.

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 A Gastropod Toxin Selectively Slows Early Transitions in the Shaker K Channel's Activation Pathway

2004 ◽  
Vol 123 (6) ◽  
pp. 685-696 ◽  
Author(s):  
Jon T. Sack ◽  
Richard W. Aldrich ◽  
William F. Gilly
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Gating Currents ◽  
K Channels ◽  
Voltage Sensors ◽  
Essentially Normal ◽  
Channel Opening ◽  
Single Channel Activity ◽  
Kinetics Of

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.

Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit

1995 ◽  
Vol 268 (3) ◽  
pp. C732-C740 ◽  
Author(s):  
A. Neely ◽  
R. Olcese ◽  
P. Baldelli ◽  
X. Wei ◽  
L. Birnbaumer ◽  
...  
Keyword(s):  
Time Course ◽  
Slow Component ◽  
Ionic Currents ◽  
Ionic Channel ◽  
Relative Increase ◽  
Beta Subunit ◽  
Single Channels ◽  
Gating Currents ◽  
Channel Opening ◽  
Dual Activation

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.

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