A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion

Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

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Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.277.5.g1055 ◽

1999 ◽

Vol 277 (5) ◽

pp. G1055-G1063 ◽

Cited By ~ 32

Author(s):

Chuanli Xu ◽

Yanjie Lu ◽

Guanghua Tang ◽

Rui Wang

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Mesenteric Artery ◽

Muscle Cells ◽

Mesenteric Arteries ◽

Delayed Rectifier ◽

Kv Channel ◽

Channel Proteins ◽

Voltage Dependent ◽

Encoding Genes

Molecular basis of native voltage-dependent K+(Kv) channels in smooth muscle cells (SMCs) from rat mesenteric arteries was investigated. The whole cell patch-clamp study revealed that a 4-aminopyridine-sensitive delayed rectifier K+ current ( I K) was the predominant K+ conductance in these cells. A systematic screening of the expression of 18 Kv channel genes using RT-PCR technique showed that six I K-encoding genes (Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2) were expressed in mesenteric artery. Although no transient outward Kv currents ( I A) were recorded in the studied SMCs, transcripts of multiple I A-encoding genes, including Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 as well as I A-facilitating Kv β-subunits (Kvβ1, Kvβ2, and Kvβ3), were detected in mesenteric arteries. Western blot analysis demonstrated that four I K-related Kv channel proteins (Kv1.2, Kv1.3, Kv1.5, and Kv2.1) were detected in mesenteric artery tissues. The presence of Kv1.2, Kv1.3, Kv1.5, and Kv2.1 channel proteins in isolated SMCs was further confirmed by immunocytochemistry study. Our results suggest that the native I K in rat mesenteric artery SMCs might be generated by heteromultimerization of Kv genes.

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Calcium-activated potassium channels from coronary smooth muscle reconstituted in lipid bilayers

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.260.6.h1779 ◽

1991 ◽

Vol 260 (6) ◽

pp. H1779-H1789 ◽

Cited By ~ 4

Author(s):

L. Toro ◽

L. Vaca ◽

E. Stefani

Keyword(s):

Smooth Muscle ◽

Lipid Bilayers ◽

Surface Membrane ◽

Dependent Manner ◽

Coronary Smooth Muscle ◽

Voltage Sensors ◽

Kca Channels ◽

Voltage Dependent ◽

Calcium Activated Potassium Channels

This work is the initial characterization of Ca(2+)-activated K+ (KCa) channels from coronary smooth muscle reconstituted into lipid bilayers. The channels were obtained from a surface membrane preparation of porcine coronary smooth muscle. KCa channels were the predominant K+ channels in this preparation. The conductance histogram (n = 137 channels) revealed two main populations of “maxi” KCa channels with conductances of 245 and 295 pS. Each population could be subdivided in two “isoforms” or “isochannels” with different functional properties (voltage and Ca2+ sensitivities and kinetics). The analysis of “burst” probability of opening showed that at pCa 4 the two isochannels of 245 pS (KCa-1 and KCa-1') had half-activation potentials (V1/2) of -80 and 6 mV, respectively. The isochannels of 295 pS (KCa-2 and KCa-2') had V1/2 of -28 and -66 mV, respectively. KCa-1 had the highest Ca2+ sensitivity; at -60 mV, the concentration of half-activation value for Ca2+ was 1.2 +/- 0.3 microM (n = 5). External tetraethylammonium reduced channel amplitude in a voltage-dependent manner; dissociation constant was 180 +/- 6 and 466 +/- 41 microM at -40 and +80 mV, respectively (n = 5). Charybdotoxin (5-50 nM) produced typical long closings. These effects were similar in all the channels. We conclude that coronary smooth muscle possesses isoforms of maxi KCa channels with Ca2+ and voltage sensors with different properties, which may confer to each channel a specific functional role.

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

The Journal of General Physiology ◽

10.1085/jgp.20028569 ◽

2002 ◽

Vol 119 (6) ◽

pp. 521-531 ◽

Cited By ~ 129

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.

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

eLife ◽

10.7554/elife.00594 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 122

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.

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Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry

Biophysical Journal ◽

10.1016/j.bpj.2019.07.011 ◽

2019 ◽

Vol 117 (4) ◽

pp. 751-766 ◽

Cited By ~ 2

Author(s):

Andrey Y. Tronin ◽

Lina J. Maciunas ◽

Kimberly C. Grasty ◽

Patrick J. Loll ◽

Haile A. Ambaye ◽

...

Keyword(s):

Neutron Interferometry ◽

Time Resolved ◽

Kv Channel ◽

Voltage Dependent

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Two-stage electro–mechanical coupling of a KV channel in voltage-dependent activation

Nature Communications ◽

10.1038/s41467-020-14406-w ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 8

Author(s):

Panpan Hou ◽

Po Wei Kang ◽

Audrey Deyawe Kongmeneck ◽

Nien-Du Yang ◽

Yongfeng Liu ◽

...

Keyword(s):

Two Stage ◽

Mechanical Coupling ◽

Kv Channel ◽

Voltage Dependent

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Constitutive Activation of the Shaker Kv Channel

The Journal of General Physiology ◽

10.1085/jgp.200308905 ◽

2003 ◽

Vol 122 (5) ◽

pp. 541-556 ◽

Cited By ~ 60

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.

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Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

The Journal of General Physiology ◽

10.1085/jgp.201411300 ◽

2015 ◽

Vol 145 (4) ◽

pp. 345-358 ◽

Cited By ~ 26

Author(s):

Itzel G. Ishida ◽

Gisela E. Rangel-Yescas ◽

Julia Carrasco-Zanini ◽

León D. Islas

Keyword(s):

Ion Channels ◽

Potassium Channel ◽

Structural Data ◽

Activation Mechanism ◽

Voltage Sensitivity ◽

Structural Interpretation ◽

Kv Channel ◽

Gating Charge ◽

Voltage Dependent ◽

Electrophysiological Findings

Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13–14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker’s, accounting for the smaller Kv1.2 gating charge.

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