scholarly journals Modification of potassium channel kinetics by amino group reagents.

1992 ◽  
Vol 99 (1) ◽  
pp. 109-129 ◽  
Author(s):  
S Spires ◽  
T Begenisich
Keyword(s):  
Channel Gating ◽  
Ionic Currents ◽  
K Channel ◽  
Delayed Rectifier ◽  
Trinitrobenzene Sulfonic Acid ◽  
Voltage Sensitivity ◽  
Amino Group ◽  
Amino Groups ◽  
Gating Currents ◽  
Simple Kinetic Model

We have examined the actions of several amino group reagents on delayed rectifier potassium channels in squid giant axons. Three general classes of reagents were used: (1) those that preserved the positive charge of amino groups; (2) those that neutralize the charge; and (3) those that replace the positive with a negative charge. All three types of reagents produced qualitatively similar effects on K channel properties. Trinitrobenzene sulfonic acid (TNBS) neutralizes the peptide terminal amino groups and the epsilon-amino group of lysine groups. TNBS (a) slowed the kinetics of macroscopic ionic currents; (b) increased the size of ionic currents at large positive voltages; (c) shifted the voltage-dependent probability of channel opening to more positive potentials but had no effect on the voltage sensitivity; and (d) altered several properties of K channel gating currents. The actions of TNBS on gating currents suggest the presence of multiple gating current components. These effects are not all coupled, suggesting that several amino groups on the external surface of K channels are important for channel gating. A simple kinetic model that considers the channel to be composed of independent heterologous subunits is consistent with most of the modifications produced by amino group reagents.

The 1997 Stevenson Award Lecture. Cardiac K+channel gating: cloned delayed rectifier mechanisms and drug modulation

1998 ◽  
Vol 76 (2) ◽  
pp. 77-89 ◽  
Author(s):  
David Fedida ◽  
Fred SP Chen ◽  
Xue Zhang
Keyword(s):  
Channel Gating ◽  
State Dependence ◽  
Structural Features ◽  
K Channel ◽  
Delayed Rectifier ◽  
Specific Reference ◽  
Channel Blockers ◽  
Antiarrhythmic Agents ◽  
Gating Currents ◽  
Voltage Gated

K+ channels are ubiquitous membrane proteins, which have a central role in the control of cell excitability. In the heart, voltage-gated delayed rectifier K+ channels, like Kv1.5, determine repolarization and the cardiac action potential plateau duration. Here we review the broader properties of cloned voltage-gated K+ channels with specific reference to the hKv1.5 channel in heart. We discuss the basic structural components of K+ channels such as the pore, voltage sensor, and fast inactivation, all of which have been extensively studied. Slow, or C-type, inactivation and the structural features that control pore opening are less well understood, although recent studies have given new insight into these problems. Information about channel transitions that occur prior to opening is provided by gating currents, which reflect charge-carrying transitions between kinetic closed states. By studying modulation of the gating properties of K+ channels by cations and with drugs, we can make a more complete interpretation of the state dependence of drug and ion interactions with the channel. In this way we can uncover the detailed mechanisms of action of K+ channel blockers such as tetraethylammonium ions and 4-aminopyridine, and antiarrhythmic agents such as nifedipine and quinidine.Key words: potassium channel, Kv1.5, channel gating, inactivation, pore region, gating currents.

Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells

1992 ◽  
Vol 263 (5) ◽  
pp. C1081-C1087 ◽  
Author(s):  
A. M. Frace ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Gh3 Cells ◽  
Disulfonic Acid ◽  
State Transitions ◽  
Trinitrobenzene Sulfonic Acid ◽  
Voltage Sensitivity ◽  
Pituitary Cells ◽  
Amino Groups ◽  
Open Probability ◽  
Long Duration

The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.

scholarly journals Activation of squid axon K+ channels. Ionic and gating current studies.

1985 ◽  
Vol 85 (4) ◽  
pp. 539-554 ◽  
Author(s):  
M M White ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
General Scheme ◽  
Channel Gating ◽  
K Channel ◽  
Na Channel ◽  
Activation Process ◽  
Gating Current ◽  
Gating Currents ◽  
Similar Time ◽  
Channel Activation

We have used data obtained from measurements of ionic and gating currents to study the process of K+ channel activation in squid giant axons. A marked improvement in the recording of K+ channel gating currents (IKg) was obtained by total replacement of Cl- in the external solution by NO-3, which eliminates approximately 50% of the Na+ channel gating current with no effect on IKg. The midpoint of the steady state charge-voltage (Qrel - V) relationship is approximately 40 mV hyperpolarized to that of the steady state activation (fo - V) curve, which is an indication that the channel has many nonconducting states. Ionic and gating currents have similar time constants for both ON and OFF pulses. This eliminates any Hodgkin-Huxley nx scheme for K+ channel activation. An interrupted pulse paradigm shows that the last step in the activation process is not rate limiting. IKg shows a nonartifactual rising phase, which indicates that the first step is either the slowest step in the activation sequence or is voltage independent. These data are consistent with the following general scheme for K+ channel activation: (formula; see text)

scholarly journals Pharmacological and kinetic analysis of K channel gating currents.

1989 ◽  
Vol 93 (2) ◽  
pp. 263-283 ◽  
Author(s):  
S Spires ◽  
T Begenisich
Keyword(s):  
Channel Gating ◽  
K Channel ◽  
Na Channel ◽  
Gating Current ◽  
Channel Conductance ◽  
Gating Currents ◽  
Current Time ◽  
Time Constants ◽  
K Channels ◽  
Low Concentrations

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.

scholarly journals Gating currents from a delayed rectifier K+ channel with altered pore structure and function

1992 ◽  
Vol 62 (1) ◽  
pp. 34-36 ◽  
Author(s):  
M. Taglialatela ◽  
G.E. Kirsch ◽  
A.M. VanDongen ◽  
J.A. Drewe ◽  
H.A. Hartmann ◽  
...  
Keyword(s):  
Pore Structure ◽  
Structure And Function ◽  
K Channel ◽  
Delayed Rectifier ◽  
Gating Currents ◽  
And Function

scholarly journals Voltage Sensitivity and Gating Charge in Shaker and Shab Family Potassium Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 723-742 ◽  
Author(s):  
Leon D. Islas ◽  
Fred J. Sigworth
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Voltage Sensitivity ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
Charge Voltage ◽  
Gating Charge ◽  
Charged Residues ◽  
Positively Charged

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, ∼13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge–voltage relationships. We find that Shab has a relatively small gating charge, ∼7.5 eo. Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 eo, essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10−9 in Shaker and below 4 × 10−8 in Kv2.1.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

Sequential gating in the human heart K+ channel Kv1.5 incorporatesQ 1 andQ 2 charge components

1999 ◽  
Vol 277 (5) ◽  
pp. H1956-H1966 ◽  
Author(s):  
J. Christian Hesketh ◽  
David Fedida
Keyword(s):  
K Channel ◽  
Delayed Rectifier ◽  
Potential Range ◽  
Charge Movement ◽  
Gating Current ◽  
Gating Currents ◽  
Charge Voltage ◽  
Hek 293 ◽  
Gating Charge ◽  
State Dependent

On-gating current from the Kv1.5 cardiac delayed rectifier K+ channel expressed in HEK-293 cells was separated into two distinct charge systems, Q 1 and Q 2, obtained from double Boltzmann fits to the charge-voltage relationship. Q 1 and Q 2 had characteristic voltage dependence and sensitivity with half-activation potentials of −29.6 ± 1.6 and −2.19 ± 2.09 mV and effective valences of 1.87 ± 0.15 and 5.53 ± 0.27 e −, respectively. The contribution to total gating charge was 0.20 ± 0.04 for Q 1 and 0.80 ± 0.04 ( n = 5) for Q 2. At intermediate depolarizations, heteromorphic gating current waveforms resulted from relatively equal contributions from Q 1 and Q 2, but with widely different kinetics. Prepulses to −20 mV moved only Q 1, simplified on-gating currents, and allowed rapid Q 2 movement. Voltage-dependent on-gating current recovery in the presence of 4-aminopyridine (1 mM) suggested a sequentially coupled movement of the two charge systems during channel activation. This allowed the construction of a linear five-state model of Q 1 and Q 2 gating charge movement, which predicted experimental on-gating currents over a wide potential range. Such models are useful in determining state-dependent mechanisms of open and closed channel block of cardiac K+ channels.

scholarly journals Ionic currents and ion channels of lobster olfactory receptor neurons.

1989 ◽  
Vol 94 (6) ◽  
pp. 1085-1099 ◽  
Author(s):  
T S McClintock ◽  
B W Ache
Keyword(s):  
Ion Channels ◽  
Olfactory Receptor ◽  
Ionic Currents ◽  
K Channel ◽  
Delayed Rectifier ◽  
Na Current ◽  
Cl Channel ◽  
Inward Currents ◽  
K Current ◽  
K Currents

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.

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