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.

Effects of K+ Channel Blockers on Developing Rat Myelinated CNS Axons: Identification of Four Types of K+Channels

2002 ◽  
Vol 87 (3) ◽  
pp. 1376-1385 ◽  
Author(s):  
Jerome Devaux ◽  
Maurice Gola ◽  
Guy Jacquet ◽  
Marcel Crest
Keyword(s):  
Optic Nerve ◽  
Refractory Period ◽  
K Channel ◽  
Channel Blockers ◽  
Optic Nerves ◽  
Kv Channel ◽  
Kv Channels ◽  
Compound Action Potentials ◽  
Channel Expression ◽  
The One

Four blockers of voltage-gated potassium channels (Kv channels) were tested on the compound action potentials (CAPs) of rat optic nerves in an attempt to determine the regulation of Kv channel expression during the process of myelination. Before myelination occurred, 4-aminopyridine (4-AP) increased the amplitude, duration, and refractory period of the CAPs. On the basis of their pharmacological sensitivity, 4-AP-sensitive channels were divided in two groups, the one sensitive to kaliotoxin (KTX), dendrotoxin-I (DTX-I), and 4-AP, and the other sensitive only to 4-AP. In addition, tetraethylammonium chloride (TEA) applied alone broadened the CAPs. At the onset of myelination, DTX-I induced a more pronounced effect than KTX; this indicates that a fourth group of channels sensitive to 4-AP and DTX-I but insensitive to KTX had developed. The effects of KTX and DTX-I gradually disappeared during the period of myelination. Electron microscope findings showed that the disappearance of these effects was correlated with the ongoing process of myelination. This was confirmed by the fact that DTX-I and KTX enlarged the CAPs of demyelinated adult optic nerves. These results show that KTX- and DTX-sensitive channels are sequestrated in paranodal regions. During the process of myelination, KTX had less pronounced effects than DTX-I on demyelinated nerves, which suggests that the density of the KTX-sensitive channels decreased during this process. By contrast, 4-AP increased the amplitude, duration, and refractory period of the CAPs at all the ages tested and to a greater extent than KTX and DTX-I. The effects of TEA alone also gradually disappeared during this period. However, effects of TEA on CAPs were observed when this substance was applied after 4-AP to the adult optic nerve; this shows that TEA-sensitive channels are not masked by the myelin sheath. In conclusion, the process of myelination seems to play an important part in the regulation and setting of Kv channels in optic nerve axons.

scholarly journals Molecular Surface of Tarantula Toxins Interacting with Voltage Sensors in Kv Channels

2004 ◽  
Vol 123 (4) ◽  
pp. 455-467 ◽  
Author(s):  
Julia M. Wang ◽  
Soung Hun Roh ◽  
Sunghwan Kim ◽  
Chul Won Lee ◽  
Jae Il Kim ◽  
...  
Keyword(s):  
Concentration Dependence ◽  
Active Surface ◽  
Molecular Surface ◽  
Aromatic Residues ◽  
Voltage Sensors ◽  
Atomic Structures ◽  
Kv Channels ◽  
Rich Diversity ◽  
Protein Toxins ◽  
Toxin Inhibition

The venom from spiders, scorpions, and sea anemone contain a rich diversity of protein toxins that interact with ion channel voltage sensors. Although atomic structures have been solved for many of these toxins, the surfaces that are critical for interacting with voltage sensors are poorly defined. Hanatoxin and SGTx are tarantula toxins that inhibit activation of Kv channels by interacting with each of the four voltage sensors. In this study we set out to identify the active surface of these toxins by alanine-scanning SGTx and characterizing the interaction of each mutant with the Kv2.1 channel. Examination of the concentration dependence for inhibition identified 15 mutants with little effect on the concentration dependence for toxin inhibition of the Kv2.1 channel, and 11 mutants that display moderate to dramatic perturbations. Mapping of these results onto the structure of SGTx identifies one face of the toxin where mutations with pronounced perturbations cluster together, and a backside of the toxin where mutations are well tolerated. The active surface of SGTx contains a ring-like assembly of highly polar residues, with two basic residues that are particularly critical, concentrically arranged around a hydrophobic protrusion containing critical aliphatic and aromatic residues. These results identify the active surface of the toxin and reveal the types of side chains that are important for interacting with voltage sensors.

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 Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Anirban Banerjee ◽  
Alice Lee ◽  
Ernest Campbell ◽  
Roderick MacKinnon
Keyword(s):  
Electron Density ◽  
Selectivity Filter ◽  
K Channel ◽  
Wild Type ◽  
Kv Channel ◽  
Pore Blocking ◽  
Kv Channels ◽  
Filter Structure ◽  
Conduction Pathway ◽  
Voltage Dependent

Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter.

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 Molecular Template for a Voltage Sensor in a Novel K+ Channel. I. Identification and Functional Characterization of KvLm, a Voltage-gated K+ Channel from Listeria monocytogenes

2006 ◽  
Vol 128 (3) ◽  
pp. 283-292 ◽  
Author(s):  
Jose S. Santos ◽  
Alicia Lundby ◽  
Cecilia Zazueta ◽  
Mauricio Montal
Keyword(s):  
Listeria Monocytogenes ◽  
Voltage Sensor ◽  
K Channel ◽  
Open Probability ◽  
Voltage Gated ◽  
Kv Channel ◽  
Kv Channels ◽  
Sensor Module ◽  
Single Channel Currents ◽  
Hallmark Feature

The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. Here we show that a novel prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes (KvLm) embodies a rudimentary, yet robust, sensor sufficient to endow it with voltage-dependent features comparable to those of eukaryotic Kv channels. The most conspicuous feature of the KvLm sequence is the nature of the sensor components: the motif is recognizable; it appears, however, to contain only three out of eight charged residues known to be conserved in eukaryotic Kv channels and accepted to be deterministic for folding and sensing. Despite the atypical sensor sequence, flux assays of KvLm reconstituted in liposomes disclosed a channel pore that is highly selective for K+ and is blocked by conventional Kv channel blockers. Single-channel currents recorded in symmetric K+ solutions from patches of enlarged Escherichia coli (spheroplasts) expressing KvLm showed that channel open probability sharply increases with depolarization, a hallmark feature of Kv channels. The identification of a voltage sensor module in KvLm with a voltage dependence comparable to that of other eukaryotic Kv channels yet encoded by a sequence that departs significantly from the consensus sequence of a eukaryotic voltage sensor establishes a molecular blueprint of a minimal sequence for a voltage sensor.

scholarly journals Tarantula Toxins Interact with Voltage Sensors within Lipid Membranes

2007 ◽  
Vol 130 (5) ◽  
pp. 497-511 ◽  
Author(s):  
Mirela Milescu ◽  
Jan Vobecky ◽  
Soung H. Roh ◽  
Sung H. Kim ◽  
Hoi J. Jung ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Membranes ◽  
Voltage Sensor ◽  
Structural Motif ◽  
Membrane Interaction ◽  
Voltage Sensors ◽  
Kv Channels ◽  
Channel Interactions ◽  
The Stability ◽  
Electrical Signaling

Voltage-activated ion channels are essential for electrical signaling, yet the mechanism of voltage sensing remains under intense investigation. The voltage-sensor paddle is a crucial structural motif in voltage-activated potassium (Kv) channels that has been proposed to move at the protein–lipid interface in response to changes in membrane voltage. Here we explore whether tarantula toxins like hanatoxin and SGTx1 inhibit Kv channels by interacting with paddle motifs within the membrane. We find that these toxins can partition into membranes under physiologically relevant conditions, but that the toxin–membrane interaction is not sufficient to inhibit Kv channels. From mutagenesis studies we identify regions of the toxin involved in binding to the paddle motif, and those important for interacting with membranes. Modification of membranes with sphingomyelinase D dramatically alters the stability of the toxin–channel complex, suggesting that tarantula toxins interact with paddle motifs within the membrane and that they are sensitive detectors of lipid–channel interactions.

scholarly journals Molecular Template for a Voltage Sensor in a Novel K+ Channel. II. Conservation of a Eukaryotic Sensor Fold in a Prokaryotic K+ Channel

2006 ◽  
Vol 128 (3) ◽  
pp. 293-300 ◽  
Author(s):  
Alicia Lundby ◽  
Jose S. Santos ◽  
Cecilia Zazueta ◽  
Mauricio Montal
Keyword(s):  
Consensus Sequence ◽  
Voltage Sensor ◽  
K Channel ◽  
Functional Coupling ◽  
Voltage Sensitivity ◽  
Conserved Residues ◽  
Kv Channel ◽  
Kv Channels ◽  
Sensor Module ◽  
The Impact

KvLm, a novel bacterial depolarization-activated K+ (Kv) channel isolated from the genome of Listeria monocytogenes, contains a voltage sensor module whose sequence deviates considerably from the consensus sequence of a Kv channel sensor in that only three out of eight conserved charged positions are present. Surprisingly, KvLm exhibits the steep dependence of the open channel probability on membrane potential that is characteristic of eukaryotic Kv channels whose sensor sequence approximates the consensus. Here we asked if the KvLm sensor shared a similar fold to that of Shaker, the archetypal eukaryotic Kv channel, by examining if interactions between conserved residues in Shaker known to mediate sensor biogenesis and function were conserved in KvLm. To this end, each of the five non-conserved residues in the KvLm sensor were mutated to their Shaker-like charged residues, and the impact of these mutations on the voltage dependence of activation was assayed by current recordings from excised membrane patches of Escherichia coli spheroplasts expressing the KvLm mutants. Conservation of pairwise interactions was investigated by comparison of the effect of single mutations to the impact of double mutations presumed to restore wild-type fold and voltage sensitivity. We observed significant functional coupling between sites known to interact in Shaker Kv channels, supporting the notion that the KvLm sensor largely retains the fold of its eukaryotic homologue.

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.

A mitochondrial uncoupler increases KCa currents but decreases KV currents in pulmonary artery myocytes

1996 ◽  
Vol 270 (1) ◽  
pp. C321-C331 ◽  
Author(s):  
X. J. Yuan ◽  
T. Sugiyama ◽  
W. F. Goldman ◽  
L. J. Rubin ◽  
M. P. Blaustein
Keyword(s):  
Pulmonary Artery ◽  
Fluorescent Imaging ◽  
K Channel ◽  
Metabolic Inhibitor ◽  
Acetoxymethyl Ester ◽  
Atp Production ◽  
Kv Channels ◽  
Kca Channels ◽  
Carbonyl Cyanide ◽  
Microscopy Techniques

Intracellular free Ca2+ concentration ([Ca2+]i) and ATP play important roles in the regulation of K- channels in pulmonary artery (PA) myocytes. Previous studies have demonstrated that hypoxia and the metabolic inhibitor, 2-deoxy-D-glucose, decrease voltage-gated K+ (KV) currents [IK(V)] and thereby depolarize PA myocytes; these effects lead to a rise in [Ca2+]i. Here, we used carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a protonophore that uncouples mitochondrial respiration from ATP production, to test whether the inhibition of oxidative phosphorylation affects K+ channel activities in rat PA myocytes. Patch-clamp and fluorescent-imaging microscopy techniques were used to measure K+ currents (IK) and [Ca2+]i, respectively. FCCP (3-5 microM) reversibly raised [Ca2-]i in the presence and absence of external Ca2+. This effect was prevented by pretreating the cells with the membrane-permeable Ca2+ chelator, 1,2-bis(2-amino-phenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). This suggests that much of the FCCP-evoked rise in [Ca2-]i was due to Ca2+ release from intracellular stores. Brief exposure to FCCP (approximately 2 min) reversibly enhanced Ik. This augmentation was not influenced by glibenclamide, an ATP-sensitive K channel blocker, but was eliminated by pretreatment with BAPTA-AM. This implies that the FCCP-evoked rise in [Ca2+]i activated Ca(2+)-activated K- (Kca) channels. Furthermore, in BAPTA-treated cells, longer application (> or = 6 min) of FCCP reversibly decreased IK(V) in PA cells bathed in Ca(2+)-free solution. These results demonstrate that FCCP affects KCa and Kv channels by different mechanisms. FCCP increases IK[Ca] by raising [Ca2+]i primarily as a result of Ca2+ release, but decreases IK(V) by a Ca(2+)-independent mechanism, presumably the inhibition of oxidative ATP production.

Sign in / Sign up

Export Citation Format

Share Document