Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

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Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

The Journal of General Physiology ◽

10.1085/jgp.201611602 ◽

2016 ◽

Vol 148 (2) ◽

pp. 119-132 ◽

Cited By ~ 15

Author(s):

Dorothy M. Kim ◽

Igor Dikiy ◽

Vikrant Upadhyay ◽

David J. Posson ◽

David Eliezer ◽

...

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Conformational Changes ◽

Ph Dependence ◽

Channel Gating ◽

Conformational Heterogeneity ◽

Ion Channel Gating ◽

Potassium Channel Kcsa ◽

Functional States ◽

Open States

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.

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Exploring the Conformational Changes Induced by Nanosecond Pulsed Electric Fields on the Voltage Sensing Domain of a Ca2+ Channel

Membranes ◽

10.3390/membranes11070473 ◽

2021 ◽

Vol 11 (7) ◽

pp. 473

Author(s):

Alvaro R. Ruiz-Fernández ◽

Leonardo Campos ◽

Felipe Villanelo ◽

Sebastian E. Gutiérrez-Maldonado ◽

Tomas Perez-Acle

Keyword(s):

Electric Field ◽

Ion Channel ◽

External Electric Field ◽

Electric Fields ◽

Conformational Changes ◽

Channel Gating ◽

Cholesterol Content ◽

Ion Channel Gating ◽

Voltage Gated ◽

Voltage Sensing Domain

Nanosecond Pulsed Electric Field (nsPEF or Nano Pulsed Stimulation, NPS) is a technology that delivers a series of pulses of high-voltage electric fields during a short period of time, in the order of nanoseconds. The main consequence of nsPEF upon cells is the formation of nanopores, which is followed by the gating of ionic channels. Literature is conclusive in that the physiological mechanisms governing ion channel gating occur in the order of milliseconds. Hence, understanding how these channels can be activated by a nsPEF would be an important step in order to conciliate fundamental biophysical knowledge with improved nsPEF applications. To get insights on both the kinetics and thermodynamics of ion channel gating induced by nsPEF, in this work, we simulated the Voltage Sensing Domain (VSD) of a voltage-gated Ca2+ channel, inserted in phospholipidic membranes with different concentrations of cholesterol. We studied the conformational changes of the VSD under a nsPEF mimicked by the application of a continuous electric field lasting 50 ns with different intensities as an approach to reveal novel mechanisms leading to ion channel gating in such short timescales. Our results show that using a membrane with high cholesterol content, under an nsPEF of 50 ns and E→ = 0.2 V/nm, the VSD undergoes major conformational changes. As a whole, our work supports the notion that membrane composition may act as an allosteric regulator, specifically cholesterol content, which is fundamental for the response of the VSD to an external electric field. Moreover, changes on the VSD structure suggest that the gating of voltage-gated Ca2+ channels by a nsPEF may be due to major conformational changes elicited in response to the external electric field. Finally, the VSD/cholesterol-bilayer under an nsPEF of 50 ns and E→ = 0.2 V/nm elicits a pore formation across the VSD suggesting a new non-reported effect of nsPEF into cells, which can be called a “protein mediated electroporation”.

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Radiolytic Footprinting Reveals Conformational Changes During Potassium Channel Gating

Biophysical Journal ◽

10.1016/j.bpj.2009.12.3819 ◽

2010 ◽

Vol 98 (3) ◽

pp. 696a

Author(s):

Sayan Gupta ◽

Rhijuta D'Mello ◽

Mark R. Chance ◽

Vassiliy N. Bavro ◽

Catherine Venien-Bryan ◽

...

Keyword(s):

Potassium Channel ◽

Conformational Changes ◽

Channel Gating ◽

Potassium Channel Gating

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Shared and unique aspects of ligand- and voltage-gated ion-channel gating

The Journal of Physiology ◽

10.1113/jp275877 ◽

2018 ◽

Vol 596 (10) ◽

pp. 1829-1832

Author(s):

Derek Bowie

Keyword(s):

Ion Channel ◽

Channel Gating ◽

Ion Channel Gating ◽

Voltage Gated

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Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-024 ◽

2009 ◽

Vol 87 (6) ◽

pp. 411-418 ◽

Cited By ~ 11

Author(s):

A.J. Horne ◽

D. Fedida

Keyword(s):

Potassium Channels ◽

Potassium Channel ◽

Voltage Clamp ◽

Channel Gating ◽

Specific Protein ◽

Channel Activation ◽

Shaker Potassium Channel ◽

Inactivation Gating ◽

Nanosecond Time Resolution ◽

Shaker Potassium Channels

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

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Evidence for a Deep Pore Activation Gate in Small Conductance Ca2+-activated K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.200709828 ◽

2007 ◽

Vol 130 (6) ◽

pp. 601-610 ◽

Cited By ~ 32

Author(s):

Andrew Bruening-Wright ◽

Wei-Sheng Lee ◽

John P. Adelman ◽

James Maylie

Keyword(s):

Cysteine Residue ◽

Selectivity Filter ◽

K Channel ◽

Homologous Region ◽

Sk Channels ◽

Voltage Gated ◽

Kv Channels ◽

Substituted Cysteine Accessibility ◽

Activation Gate ◽

Small Conductance

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (Kv) channels, but are distinct in that they are gated solely by calcium (Ca2+), not voltage. For Kv channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd2+) and barium (Ba2+), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd2+ applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd2+ and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba2+ applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba2+ is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba2+ pre-block slows MTSEA modification of A384C in open but not in closed (Ba2+-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.

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The Polar T1 Interface Is Linked to Conformational Changes that Open the Voltage-Gated Potassium Channel

Cell ◽

10.1016/s0092-8674(00)00088-x ◽

2000 ◽

Vol 102 (5) ◽

pp. 657-670 ◽

Cited By ~ 129

Author(s):

Daniel L Minor ◽

Yu-Fung Lin ◽

Bret C Mobley ◽

Abigail Avelar ◽

Yuh Nung Jan ◽

...

Keyword(s):

Potassium Channel ◽

Conformational Changes ◽

Voltage Gated

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Structural and molecular insight into the pH-induced low-permeability of the voltage-gated potassium channel Kv1.2 through dewetting of the water cavity

10.1101/775759 ◽

2019 ◽

Author(s):

Juhwan Lee ◽

Mooseok Kang ◽

Sangyeol Kim ◽

Iksoo Chang

Keyword(s):

Ion Channels ◽

Ion Channel ◽

Potassium Ion ◽

Low Permeability ◽

Voltage Gated ◽

Kv Channels ◽

Proton Concentration ◽

Electrical Voltage ◽

Gating Mechanism ◽

Ph Dependent

AbstractUnderstanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1–S4 helices) and central pore domains (S5–S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.Author SummaryThe acid sensing ion channels are a biological machinery for maintaining the cell functional under the acidic or basic cellular environment. Understanding the pH-dependent gating mechanism of such channels provides the structural insight to design the molecular strategy in regulating the acidosis. Here, we studied the voltage-gated potassium ion channel Kv1.2 which senses not only the electrical voltage across the channels but also the cellular acidity. We uncovered that two key residues E327 and H418 in the pore domain of Kv1.2 channel play a role as pH-switch in that their protonation control the gating of the pore in Kv1.2 channel. It offered a molecular insight how the acidity reduces the ion permeability in voltage-gated potassium channels.

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