scholarly journals Constitutive Activation of the Shaker Kv Channel

2003 ◽  
Vol 122 (5) ◽  
pp. 541-556 ◽  
Author(s):  
Manana Sukhareva ◽  
David H. Hackos ◽  
Kenton J. Swartz
Keyword(s):  
Single Channel ◽  
Membrane Depolarization ◽  
Ion Conduction ◽  
K Channels ◽  
Voltage Sensors ◽  
Kv Channel ◽  
Kv Channels ◽  
Activation Gate ◽  
Sensor Activation ◽  
Primary Activation

In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521–532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.

scholarly journals Stabilizing the Closed S6 Gate in the Shaker K v Channel Through Modification of a Hydrophobic Seal

2004 ◽  
Vol 124 (4) ◽  
pp. 319-332 ◽  
Author(s):  
Tetsuya Kitaguchi ◽  
Manana Sukhareva ◽  
Kenton J. Swartz
Keyword(s):  
Double Mutant ◽  
Single Channel ◽  
Ion Permeation ◽  
Ion Conduction ◽  
K Channels ◽  
Voltage Sensors ◽  
Gating Charge ◽  
Extracellular Region ◽  
Activation Gate ◽  
Primary Activation

The primary activation gate in K+ channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker (Hackos et al., 2002), we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K+ channels.

The role of K-channels in the formation of a nociceptive signal in the rat trigeminal nerve

2021 ◽  
Author(s):  
A.D. Buglinina ◽  
T.M. Verkhoturova ◽  
O.Sh. Gafurov ◽  
K.S. Koroleva ◽  
G.F. Sitdikova
Keyword(s):  
Key Words ◽  
Trigeminal Nerve ◽  
Key Words Migraine ◽  
K Channels ◽  
Kv Channel ◽  
Inflammatory Agent ◽  
Kv Channels ◽  
Pain Signal ◽  
Isolated Rat

The central problem of this work is to elucidate the mechanisms of pain in migraine and to establish the role of Kv channels in regulating the excitability of meningeal afferents of the trigeminal nerve that form a pain signal in migraine. The study was conducted on a preparation of an isolated rat skull. It was found that Kv-channel inhibitors 4-aminopyridine (100 microns and 1 mM) and tetraethylammonium (5mm) lead to an increase in the excitability of trigeminal nerve afferents, at the same time, this effect was partially removed by a nonsteroidal anti–inflammatory agent - naproxen, and was not sensitive to sumatriptan, a classic anti-migraine drug. Key words: migraine, K-channels, trigeminal nerve, 4-aminopyridine, tetraethylammonium, naproxen, sumatriptan.

scholarly journals Scanning the Intracellular S6 Activation Gate in the Shaker K+ Channel

2002 ◽  
Vol 119 (6) ◽  
pp. 521-531 ◽  
Author(s):  
David H. Hackos ◽  
Tsg-Hui Chang ◽  
Kenton J. Swartz
Keyword(s):  
Ionic Currents ◽  
K Channel ◽  
Dependent Manner ◽  
Kv Channel ◽  
Closed State ◽  
Kv Channels ◽  
Gate Structure ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Gate Region

In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed 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.

scholarly journals Coupling between Voltage Sensors and Activation Gate in Voltage-gated K+ Channels

2002 ◽  
Vol 120 (5) ◽  
pp. 663-676 ◽  
Author(s):  
Zhe Lu ◽  
Angela M. Klem ◽  
Yajamana Ramu
Keyword(s):  
Electrical Signal ◽  
K Channel ◽  
Coupling Mechanism ◽  
Excitable Cells ◽  
Voltage Sensing ◽  
K Channels ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Activation Gate

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.

scholarly journals Gating of O2-sensitive K+ channels of arterial chemoreceptor cells and kinetic modifications induced by low PO2.

1992 ◽  
Vol 100 (3) ◽  
pp. 427-455 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Kinetic Scheme ◽  
Membrane Depolarization ◽  
Kinetic Properties ◽  
K Channels ◽  
Glomus Cells ◽  
Activation Pathway ◽  
Closed State

We have studied the kinetic properties of the O2-sensitive K+ channels (KO2 channels) of dissociated glomus cells from rabbit carotid bodies exposed to variable O2 tension (PO2). Experiments were done using single-channel and whole-cell recording techniques. The major gating properties of KO2 channels in excised membrane patches can be explained by a minimal kinetic scheme that includes several closed states (C0 to C4), an open state (O), and two inactivated states (I0 and I1). At negative membrane potentials most channels are distributed between the left-most closed states (C0 and C1), but membrane depolarization displaces the equilibrium toward the open state. After opening, channels undergo reversible transitions to a short-living closed state (C4). These transitions configure a burst, which terminates by channels either returning to a closed state in the activation pathway (C3) or entering a reversible inactivated conformation (I0). Burst duration increases with membrane depolarization. During a maintained depolarization, KO2 channels make several bursts before ending at a nonreversible, absorbing, inactivated state (I1). On moderate depolarizations, KO2 channels inactivate very often from a closed state. Exposure to low PO2 reversibly induces an increase in the first latency, a decrease in the number of bursts per trace, and a higher occurrence of closed-state inactivation. The open state and the transitions to adjacent closed or inactivated states seem to be unaltered by hypoxia. Thus, at low PO2 the number of channels that open in response to a depolarization decreases, and those channels that follow the activation pathway open more slowly and inactivate faster. At the macroscopic level, these changes are paralleled by a reduction in the peak current amplitude, slowing down of the activation kinetics, and acceleration of the inactivation time course. The effects of low PO2 can be explained by assuming that under this condition the closed state C0 is stabilized and the transitions to the absorbing inactivated state I1 are favored. The fact that hypoxia modifies kinetically defined conformational states of the channels suggests that O2 levels determine the structure of specific domains of the KO2 channel molecule. These results help to understand the molecular mechanisms underlying the enhancement of the excitability of glomus cells in response to hypoxia.

scholarly journals Opening the Shaker K+ channel with hanatoxin

2013 ◽  
Vol 141 (2) ◽  
pp. 203-216 ◽  
Author(s):  
Mirela Milescu ◽  
Hwa C. Lee ◽  
Chan Hyung Bae ◽  
Jae Il Kim ◽  
Kenton J. Swartz
Keyword(s):  
K Channel ◽  
Voltage Sensors ◽  
Channel Interaction ◽  
Kv Channel ◽  
Kv Channels ◽  
Structural Details ◽  
In The Wild ◽  
Protein Toxins ◽  
Electrical Signaling ◽  
Voltage Relationship

Voltage-activated ion channels open and close in response to changes in membrane voltage, a property that is fundamental to the roles of these channels in electrical signaling. Protein toxins from venomous organisms commonly target the S1–S4 voltage-sensing domains in these channels and modify their gating properties. Studies on the interaction of hanatoxin with the Kv2.1 channel show that this tarantula toxin interacts with the S1–S4 domain and inhibits opening by stabilizing a closed state. Here we investigated the interaction of hanatoxin with the Shaker Kv channel, a voltage-activated channel that has been extensively studied with biophysical approaches. In contrast to what is observed in the Kv2.1 channel, we find that hanatoxin shifts the conductance–voltage relation to negative voltages, making it easier to open the channel with membrane depolarization. Although these actions of the toxin are subtle in the wild-type channel, strengthening the toxin–channel interaction with mutations in the S3b helix of the S1-S4 domain enhances toxin affinity and causes large shifts in the conductance–voltage relationship. Using a range of previously characterized mutants of the Shaker Kv channel, we find that hanatoxin stabilizes an activated conformation of the voltage sensors, in addition to promoting opening through an effect on the final opening transition. Chimeras in which S3b–S4 paddle motifs are transferred between Kv2.1 and Shaker Kv channels, as well as experiments with the related tarantula toxin GxTx-1E, lead us to conclude that the actions of tarantula toxins are not simply a product of where they bind to the channel, but that fine structural details of the toxin–channel interface determine whether a toxin is an inhibitor or opener.

scholarly journals Trapping of a Methanesulfonanilide by Closure of the Herg Potassium Channel Activation Gate

2000 ◽  
Vol 115 (3) ◽  
pp. 229-240 ◽  
Author(s):  
John S. Mitcheson ◽  
Jun Chen ◽  
Michael C. Sanguinetti
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Membrane Hyperpolarization ◽  
K Channels ◽  
Large Size ◽  
Channel Opening ◽  
Activated State ◽  
Activation Gate ◽  
Herg Channels ◽  
Positively Charged

Deactivation of voltage-gated potassium (K+) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC50 = 32 nM) of HERG K+ channels. This bulky compound (7 × 20 Å) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 μM MK-499 to occur with time constants of 10.5 and 52.7 s at −160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K+ channel.

scholarly journals Molecular Template for a Voltage Sensor in a Novel K+ Channel. III. Functional Reconstitution of a Sensorless Pore Module from a Prokaryotic Kv Channel

2008 ◽  
Vol 132 (6) ◽  
pp. 651-666 ◽  
Author(s):  
Jose S. Santos ◽  
Sergey M. Grigoriev ◽  
Mauricio Montal
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Module ◽  
Ion Selectivity ◽  
Ionic Currents ◽  
K Channel ◽  
Voltage Sensing ◽  
Kv Channel ◽  
Kv Channels ◽  
Conduction Properties

KvLm is a prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue–specific contributions to Kv conduction properties.

scholarly journals Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel

2013 ◽  
Vol 288 (23) ◽  
pp. 16619-16628 ◽  
Author(s):  
Jose S. Santos ◽  
Ruhma Syeda ◽  
Mauricio Montal
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Single Channel ◽  
Cysteine Residue ◽  
Covalent Attachment ◽  
Voltage Sensitivity ◽  
Voltage Gated ◽  
Kv Channel ◽  
Kv Channels ◽  
Peptide Toxin

Voltage-gated K+ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K+ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K+ with unchanged fidelity. The implication is that the voltage sensitivity of K+ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.

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