scholarly journals Voltage-dependent conformational changes in human Ca2+- and voltage-activated K+ channel, revealed by voltage-clamp fluorometry

2006 ◽  
Vol 103 (33) ◽  
pp. 12619-12624 ◽  
Author(s):  
N. Savalli ◽  
A. Kondratiev ◽  
L. Toro ◽  
R. Olcese
Keyword(s):  
Voltage Clamp ◽  
Conformational Changes ◽  
K Channel ◽  
Voltage Dependent

Diverse current and voltage responses to baclofen in an identified molluscan photoreceptor

1995 ◽  
Vol 74 (2) ◽  
pp. 506-518 ◽  
Author(s):  
L. D. Matzel ◽  
I. A. Muzzio ◽  
R. F. Rogers
Keyword(s):  
Voltage Clamp ◽  
Action Potentials ◽  
Gabab Receptor ◽  
Outward Current ◽  
Gabab Receptors ◽  
Equilibrium Potential ◽  
K Channel ◽  
Electrode Voltage ◽  
Voltage Dependent ◽  
K Current

1. gamma-Aminobuturic acid-B (GABAB) receptors play a role in the mediation of slow inhibitory postsynaptic potentials in mammalian as well as some nonmammalian species. In identified photoreceptors from the marine mollusc Hermissenda, recent evidence has suggested that GABA, as well as the GABAB receptor agonist baclofen, might simultaneously modulate multiple conductances on the postsynaptic membrane. Here, using intracellular current-clamp and single-electrode voltage-clamp techniques, we have characterized responses to baclofen in the B photoreceptors of the Hermissenda eye. 2. Microapplication of baclofen (12.5–62.5 microM) to the terminal branches of the B photoreceptors induced a slow, concentration-dependent hyperpolarization (approximately 3–8 mV) that was accompanied by a cessation of spontaneous action potentials and a positive shift in firing threshold. Both the hyperpolarization and the shift in spike threshold in response to baclofen were attenuated largely by the K+ channel blocker tetraethylammonium chloride (TEA; 50 mM). 3. Bath application of baclofen (100 microM) decreased the amplitude, duration, and the afterhyperpolarization (AHP) of evoked action potentials. Although baclofen's effect on spike duration and amplitude persisted in the absence of extracellular Ca2+, the reduction of the AHP by baclofen was eliminated, suggesting that multiple conductances mediated the baclofen-induced modification of the action potential. 4. Using a single-electrode voltage-clamp technique, microapplication of baclofen to the terminal branches of the B photoreceptor produced a slow, net outward current (< 0.5 nA) that reversed near the equilibrium potential for K+ and shifted to more positive potentials when extracellular K+ was increased, in approximate agreement with the Nernst equation for K+. 5. Baclofen induced an increase in amplitude of the nonvoltage dependent leak conductance (IL), and the increase was blocked by TEA. The baclofen-induced increase of IL was accompanied by an increase in amplitude and a negative shift in the voltage dependence of a slow, steeply voltage-dependent K+ current (IK), which displays selective sensitivity to TEA but does not normally contribute to leak conductance. The amplitude and steady-state inactivation of a fast, transient K+ current, as well as the amplitude of an inwardly rectifying K+ current were unaffected by baclofen. 6. Both the rate of activation as well as the amplitude of a voltage-dependent Ca2+ current (ICa) were reduced by baclofen. The reduction of ICa resulted in a concomitant suppression of a Ca(2+)-dependent K+ current (IK-Ca) that was sufficient to account for the reduction of the AHP after evoked action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

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

2007 ◽  
Vol 35 (5) ◽  
pp. 1080-1082 ◽  
Author(s):  
T.W. Claydon ◽  
D. Fedida
Keyword(s):  
Ion Channel ◽  
Potassium Channel ◽  
Real Time ◽  
Voltage Clamp ◽  
Conformational Changes ◽  
Channel Gating ◽  
K Channel ◽  
Voltage Gated ◽  
Kv Channels ◽  
Health And Disease

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.

scholarly journals Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

2006 ◽  
Vol 127 (5) ◽  
pp. 539-555 ◽  
Author(s):  
Leila V. Virkki ◽  
Heini Murer ◽  
Ian C. Forster
Keyword(s):  
Voltage Clamp ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Type Ii ◽  
New Model ◽  
Fluorescence Measurements ◽  
Voltage Dependent ◽  
Electrophysiological Studies ◽  
Ion Substitution ◽  
Charge Movements

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

scholarly journals Voltage-Dependent Structural Interactions in the Shaker K+ Channel

2000 ◽  
Vol 115 (2) ◽  
pp. 123-138 ◽  
Author(s):  
Seema K. Tiwari-Woodruff ◽  
Meng-chin A. Lin ◽  
Christine T. Schulteis ◽  
Diane M. Papazian
Keyword(s):  
Conformational Changes ◽  
Voltage Sensor ◽  
K Channel ◽  
Charge Reversal ◽  
K Channels ◽  
Closed Conformation ◽  
Preliminary Model ◽  
Structural Interactions ◽  
Voltage Dependent ◽  
Double Charge

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K+ channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys. J. 72:1489–1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K+ channels.

scholarly journals Proton wires mediate the optical signal for ArcLight-type Genetically Encoded Voltage Indicators

2020 ◽  
Author(s):  
B.E Kang ◽  
L. M. Leong ◽  
Y. Kim ◽  
K. Miyazaki ◽  
W. N. Ross ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Negative Charge ◽  
Random Mutagenesis ◽  
Cell Voltage ◽  
Optical Signal ◽  
Excitation Wavelength ◽  
Optical Signals ◽  
Voltage Dependent

AbstractThe genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of ‘hot spots’ capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the β-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp.Statement of SignificanceArcLight-type GEVIs utilize proton pathways that send charge information outside of the FP to the internal chromophore enabling voltage induced conformational changes to affect fluorescence. These pathways are excitation wavelength specific suggesting that different external positions affect the protonated and deprotonated states of the chromophore.

scholarly journals Structural Implications of Fluorescence Quenching in the Shaker K+ Channel

1998 ◽  
Vol 112 (4) ◽  
pp. 391-408 ◽  
Author(s):  
Albert Cha ◽  
Francisco Bezanilla
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
The State ◽  
K Channel ◽  
Ph Titration ◽  
Shaker Potassium Channel ◽  
Channel Opening ◽  
Nearby Region ◽  
Voltage Dependent ◽  
S4 Segment

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.

scholarly journals Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons.

1981 ◽  
Vol 78 (1) ◽  
pp. 43-61 ◽  
Author(s):  
I Inoue
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Voltage Clamp ◽  
Time Constant ◽  
Equilibrium Potential ◽  
Giant Axons ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Time Courses ◽  
Calcium Permeability

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.

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