scholarly journals Voltage-dependent Membrane Displacements Measured by Atomic Force Microscopy

1998 ◽  
Vol 111 (1) ◽  
pp. 65-74 ◽  
Author(s):  
J. Mosbacher ◽  
M. Langer ◽  
J.K.H. Hörber ◽  
F. Sachs
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Electromechanical Coupling ◽  
Scanning Force Microscopy ◽  
Hek293 Cells ◽  
Displacement Current ◽  
K Channels ◽  
Force Microscopy ◽  
Flexoelectric Effect ◽  
Voltage Dependent

Cells use polar molecules in the membrane to sense changes in the transmembrane potential. The opening of voltage-gated ion channels and membrane bending due to the inverse flexoelectric effect are two examples of such electromechanical coupling. We have looked for membrane motions in an electric field using atomic (or scanning) force microscopy (AFM) with the intent of studying voltage-dependent conformational changes of ion channels. Voltage-clamped HEK293 cells were either untransfected controls or transfected with Shaker K+ channels. Using a ± 10-mV peak–peak AC carrier stimulus, untransfected cells moved 0.5–15 nm normal to the plane of the membrane. These movements tracked the voltage at frequencies >1 kHz with a phase lead of 60–120°, as expected of a displacement current. The movement was outward with depolarization, but the holding potential only weakly influenced the amplitude of the movement. In contrast, cells transfected with a noninactivating mutant of Shaker K+channels showed similar movements, but these were sensitive to the holding potential; decreasing with depolarization between −80 and 0 mV. Searching for artifactual origins of these movements, we used open or sealed pipettes and AFM cantilever placements just above the cells. These results were negative, suggesting that the observed movements were produced by the cell membrane rather than by movement of the patch pipette, or by acoustic or electrical interactions of the membrane with the AFM tip. In control cells, the electrical motor may arise from the flexoelectric effect, where changes in potential induce changes in curvature. In transfected cells, it appears that channel-specific movements also occurred. These experiments demonstrate that the AFM may be able to exploit voltage-dependent movements as a source of contrast for imaging membrane components. The electrically induced motility will cause twitching during action potentials, and may have physiological consequences.

Uterine Excitability and Ion Channels and Their Changes with Gestation and Hormonal Environment

2020 ◽  
Vol 83 (1) ◽  
Author(s):  
Susan Wray ◽  
Sarah Arrowsmith
Keyword(s):  
Ion Channels ◽  
Inward Rectifier ◽  
Cation Channels ◽  
Annual Review ◽  
Publication Date ◽  
K Channels ◽  
Voltage Gated ◽  
New Findings ◽  
Voltage Dependent ◽  
Pore Domain

We address advances in the understanding of myometrial physiology, focusing on excitation and the effects of gestation on ion channels and their relevance to labor. This review moves through pioneering studies to exciting new findings. We begin with the myometrium and its myocytes and describe how excitation might initiate and spread in this myogenic smooth muscle. We then review each of the ion channels in the myometrium: L- and T-type Ca2+ channels, KATP (Kir6) channels, voltage-dependent K channels (Kv4, Kv7, and Kv11), twin-pore domain K channels (TASK, TREK), inward rectifier Kir7.1, Ca2+-activated K+ channels with large (KCNMA1, Slo1), small (KCNN1–3), and intermediate (KCNN4) conductance, Na-activated K channels (Slo2), voltage-gated (SCN) Na+ and Na+ leak channels, nonselective (NALCN) channels, the Na K-ATPase, and hyperpolarization-activated cation channels. We finish by assessing how three key hormones— oxytocin, estrogen, and progesterone—modulate and integrate excitability throughout gestation. Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

2014 ◽  
Vol 144 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Benjamin M. Haehnel ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Electromechanical Coupling ◽  
Structural Integrity ◽  
Voltage Sensor ◽  
Amino Acid Residues ◽  
Kv Channel ◽  
Electromechanical Transduction ◽  
Voltage Dependent ◽  
Graph Theoretical Approach

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.

Scanning Force Microscopy and Nanomangulation: Studies of Dna and Proteins Involved in Dna Repair

1999 ◽  
Vol 5 (S2) ◽  
pp. 1004-1005
Author(s):  
Dorothy Erie ◽  
Glenn Ratcliff ◽  
Martin Guthold ◽  
Valerie Bullock ◽  
Michelle Pliske ◽  
...  
Keyword(s):  
Dna Repair ◽  
Physical Properties ◽  
Conformational Changes ◽  
Excision Repair ◽  
Protein Complexes ◽  
Scanning Force Microscopy ◽  
Force Microscopy ◽  
Scanning Force ◽  
Base Excision ◽  
User Friendly

Repair of damaged or incorrectly matched DNA is essential to the survival of all organisms. Consequently cells have devised a plentitude of pathways for repair. We have been investigating the mechanisms of mismatch repair and base excision repair. Both of these repair processes involve a large number of proteins that interact with one another as well as with DNA. Our long-term goal is to assemble complexes that are fully functional for DNA repair and to image the process of DNA repair. In addition, we wish to i) determine the stoicheometry of binding of the protein complexes to each other and to DNA, ii) monitor conformational changes due to substrate binding, iii) measure physical properties of DNA and the complexes. To accomplish this end, we have endeavored to improve techniques for solution imaging as well as those for data analysis. In this presentation I will discuss data on the stoicheometry of binding in several protein complexes and data on the physical properties of DNA.To measure the physical properties of DNA, we utilize a nanoManipulator, a modified Scanning Force Microscope with a novel, user-friendly interface that allows easy and controlled manipulation of nanometer-sized samples.

scholarly journals Dihydropyridine Action on Voltage-dependent Potassium Channels Expressed in Xenopus Oocytes

1997 ◽  
Vol 109 (2) ◽  
pp. 169-180 ◽  
Author(s):  
Vladimir Avdonin ◽  
Erwin F. Shibata ◽  
Toshinori Hoshi
Keyword(s):  
Ion Channels ◽  
Potassium Channels ◽  
Xenopus Oocytes ◽  
K Channels ◽  
Open Time ◽  
Voltage Dependent ◽  
Ca2 Channels

Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca2+ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K+ channels. We examined the effects of DHPs on the Shaker K+ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K+ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.

scholarly journals Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

2018 ◽  
Vol 115 (34) ◽  
pp. E8086-E8095 ◽  
Author(s):  
Galen E. Flynn ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Cyclic Nucleotide ◽  
Electromechanical Coupling ◽  
Canonical Model ◽  
Hcn Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Pore Domain

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5N-term region is involved in VSD–PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD–PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.

scholarly journals Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

2004 ◽  
Vol 166 (7) ◽  
pp. 1003-1014 ◽  
Author(s):  
Gideon Lansbergen ◽  
Yulia Komarova ◽  
Mauro Modesti ◽  
Claire Wyman ◽  
Casper C. Hoogenraad ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Binding Motif ◽  
Force Microscopy ◽  
Scanning Force ◽  
Metal Binding Motif

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

scholarly journals Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

2005 ◽  
Vol 126 (5) ◽  
pp. 419-428 ◽  
Author(s):  
Donato del Camino ◽  
Max Kanevsky ◽  
Gary Yellen
Keyword(s):  
Conformational Changes ◽  
Charge Movement ◽  
Ion Flow ◽  
K Channels ◽  
Closed State ◽  
Channel Opening ◽  
Activated State ◽  
Form Part ◽  
Voltage Dependent ◽  
Flow Through

Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

scholarly journals Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Xiao Tao ◽  
Roderick MacKinnon
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Voltage Sensor ◽  
Structural Domains ◽  
Structural Class ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels ◽  
Gated Ion Channels

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

scholarly journals AKT2/3 Subunits Render Guard Cell K+ Channels Ca2+ Sensitive

2005 ◽  
Vol 125 (5) ◽  
pp. 483-492 ◽  
Author(s):  
Natalya Ivashikina ◽  
Rosalia Deeken ◽  
Susanne Fischer ◽  
Peter Ache ◽  
Rainer Hedrich
Keyword(s):  
Guard Cell ◽  
Guard Cells ◽  
Inward Rectifier ◽  
Hek293 Cells ◽  
K Channel ◽  
Dependent Manner ◽  
Wild Type ◽  
K Channels ◽  
Isolation And Characterization ◽  
Voltage Dependent

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.

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