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 Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367.

1982 ◽  
Vol 80 (2) ◽  
pp. 203-230 ◽  
Author(s):  
J A Heiny ◽  
J Vergara
Keyword(s):  
Skeletal Muscle ◽  
Voltage Clamp ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Optical Signal ◽  
Intensity Change ◽  
Optical Signals ◽  
Potential Control ◽  
Skeletal Muscle Fibers ◽  
T System

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.

Oxytocin actions on voltage-dependent ionic channels in pregnant rat uterine smooth muscle cells

1992 ◽  
Vol 70 (12) ◽  
pp. 1597-1603 ◽  
Author(s):  
Yoshihito Inoue ◽  
Keiichi Shimamura ◽  
Nicholas Sperelakis
Keyword(s):  
Smooth Muscle ◽  
Voltage Clamp ◽  
Calcium Current ◽  
Uterine Contraction ◽  
Cell Voltage ◽  
Ionic Channels ◽  
Pregnant Rat ◽  
Uterine Smooth Muscle ◽  
Voltage Dependent ◽  
Sodium And Potassium

The effects of oxytocin, a uterotonic polypeptide hormone, on the voltage-dependent slow calcium, fast sodium, and potassium channel currents were studied using whole-cell voltage clamp of freshly isolated cells from late pregnant (18–21 day) rat myometrium. The calcium current was rapidly inhibited by oxytocin (about 25% inhibition at 20 nM) in a dose-dependent manner, and this inhibitory effect was completely reversible by washout. However, inhibition was not observed when barium was used as the charge carrier. Sodium current and potassium current were not modified by oxytocin, thus sodium and potassium currents may not play important roles in oxytocin-induced augmentation of uterine contraction. It is concluded that oxytocin stimulates uterine contraction by mechanisms other than augmentation of the voltage-dependent calcium current, e.g., by release of Ca from sarcoplasmic reticulum (by inositol trisphosphate) or by activation of a receptor-operated Ca channel. The inhibition of the slow calcium current may be induced by the elevation of [Ca]i.Key words: oxytocin, ionic channels, uterine smooth muscle, whole-cell voltage clamp, pregnant rat myometrium.

Protein kinase C stimulates Ca2+ current in pregnant rat myometrial cells

1994 ◽  
Vol 72 (11) ◽  
pp. 1304-1307 ◽  
Author(s):  
Keiichi Shimamura ◽  
Masumi Kusaka ◽  
Nicholas Sperelakis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Myometrial Cells ◽  
Kinase C ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent

The factors that regulate the voltage-dependent Ca2+ channels in pregnant uterine smooth muscle cells have not been elucidated, including any roles for protein kinase C (PKC). Therefore, the role of PKC in the regulation of the slow (L type) Ca2+ channels was examined in myometrial cells isolated from late pregnant (18–19 day) rat uterus, using the nystatin-perforated whole-cell voltage clamp. A PKC activator, phorbol 12, 13-dibutyrate (PDB), increased the L-type Ca2+ current (ICa(L)). Bath application of PDB (0.03 and 0.3 μM) increased the peak amplitude of ICa(L) by 21 ± 14% (n = 6) and 37 ± 8% (n = 9, p < 0.01), respectively. PDB did not change the holding current or shift the current–voltage relationship for ICa(L). The PKC inhibitors, H-7 (20 μM) or staurosporine (10 nM), reversed the effect of PDB. These results indicate that PKC may play a role in regulating Ca2+ channel function in pregnant rat myometrial cells and, therefore, may be involved in control of uterine contraction.Key words: protein kinase C, phorbol ester, calcium current, myometrial cell, nystatin-perforated patch, whole-cell voltage clamp.

Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells

1989 ◽  
Vol 256 (5) ◽  
pp. H1478-H1492 ◽  
Author(s):  
Y. Hirano ◽  
H. A. Fozzard ◽  
C. T. January
Keyword(s):  
Kinetic Parameters ◽  
Voltage Clamp ◽  
Purkinje Cells ◽  
Current Density ◽  
Voltage Dependence ◽  
Cell Voltage ◽  
Voltage Clamp Technique ◽  
Recovery From Inactivation ◽  
Cardiac Purkinje Cells ◽  
Voltage Dependent

Two types of Ca2+ currents were recorded in single dialyzed canine cardiac Purkinje cells using a whole cell voltage clamp technique. T-type current was easily separated from L-type current, because its voltage dependence of inactivation and activation was more negative and it decayed rapidly. L-type current was available at more depolarized holding potentials, activated at more positive voltages, and decayed slowly. In 2 mM extracellular Ca2+ concentration [( Ca]o), the average peak T- and L-type current density was 1.70 and 2.87 pA/pF, respectively. T-type current was relatively insensitive to modification by Ca2+, nifedipine, Cd2+, BAY K 8644, or isoproterenol. T-type current was more sensitive to block by Ni2+ and amiloride. Replacement of Ca2+ by Ba2+ or Sr2+ did not increase T-type current. Changes in the Ca2+ or Ba2+ concentration caused parallel shifts in the voltage dependence of several kinetic parameters for L- and T-type current. In 2 mM [Ca]o, the V1/2 (Boltzmann fit) for inactivation of T-type current was -68 mV with a slope of 3.9, and for L-type current the V1/2 was -31 mV with a slope of 5.5. Recovery from inactivation of L- and T-type current was voltage dependent, and for similar conditions L-type current recovered from inactivation more rapidly than T-type current. These findings show that T- and L-type currents are large in cardiac Purkinje cells, and they can easily be separated by their voltage, kinetic, and pharmacological differences. Both may have important physiological roles.

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.

Oxytocin induces an inward current in pregnant rat myometrial cells

1994 ◽  
Vol 72 (7) ◽  
pp. 759-763 ◽  
Author(s):  
Keiichi Shimamura ◽  
Masumi Kusaka ◽  
Nicholas Sperelakis
Keyword(s):  
Smooth Muscle ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Induced Current ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Inward Current ◽  
Uterine Smooth Muscle ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent

The effects of oxytocin (OT) on holding current were studied in uterine smooth muscle cells freshly isolated from the longitudinal layer of 18–20 day pregnant rats, using the nystatin method of whole-cell voltage clamp. As we previously reported, the voltage-dependent Ca2+ current (L type) was partially inhibited by OT (about 30% inhibition at 1 μM). When the cells were held at the holding potential (HP) of −60 mV and the holding current was monitored, OT induced an inward current. The amplitude of this OT-induced current was 72 ± 26 pA (n = 27). When the cell was held at more positive potentials (HP 0 or +40 mV), the OT-induced current reversed direction, becoming outward. This current usually was long lasting (74% of cells responding to OT); a transient current was observed in 26% of the cells. In the absence of either Na+ or Ca2+ in the bath solution, OT induced an inward current (at HP −60 mV). However, the OT-induced current was absent when both of these ions were omitted from the bath. These results suggest that OT induces an inward current through receptor-activated nonselective cation channels. The resulting increase of intracellular Ca2+ may contribute to the inhibition of voltage-dependent Ca2+ current produced by OT. This OT-induced current may also play an important role for membrane depolarization and accompanying contraction produced by OT in pregnant rat myometrium.Key words: oxytocin receptor activated channel, uterine smooth muscle, pregnant rat myometrium, holding current, whole-cell voltage clamp.

A model of the action potential and underlying membrane currents in a rabbit atrial cell

1996 ◽  
Vol 271 (4) ◽  
pp. H1666-H1696 ◽  
Author(s):  
D. S. Lindblad ◽  
C. R. Murphey ◽  
J. W. Clark ◽  
W. R. Giles
Keyword(s):  
Action Potential ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Ionic Currents ◽  
Membrane Currents ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent ◽  
Atrial Myocyte ◽  
Atrial Cell

We have developed a mathematical model of the rabbit atrial myocyte and have used it in an examination of the ionic basis of the atrial action potential. Available biophysical data have been incorporated into the model to quantify the specific ultrastructural morphology, intracellular ion buffering, and time- and voltage-dependent currents and transport mechanisms of the rabbit atrial cell. When possible, mathematical expressions describing ionic currents identified in rabbit atrium are based on whole cell voltage-clamp data from enzymatically isolated rabbit atrial myocytes. This membrane model is coupled to equations describing Na+, K+, and Ca2+ homeostasis, including the uptake and release of Ca2+ by the sarcoplasmic reticulum and Ca2+ buffering. The resulting formulation can accurately simulate the whole cell voltage-clamp data on which it is based and provides fits to a family of rabbit atrial cell action potentials obtained at 35 degrees C over a range of stimulus rates (0.2–3.0 Hz). The model is utilized to provide a qualitative prediction of the intracellular Ca2+ concentration transient during the action potential and to illustrate the interactions between membrane currents that underlie repolarization in the rabbit atrial myocyte.

Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells

1992 ◽  
Vol 262 (3) ◽  
pp. C598-C606 ◽  
Author(s):  
S. J. Quinn ◽  
U. Brauneis ◽  
D. L. Tillotson ◽  
M. C. Cornwall ◽  
G. H. Williams
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Cytosolic Calcium ◽  
Zona Glomerulosa ◽  
Voltage Dependent ◽  
Low Threshold ◽  
K Concentration ◽  
Ca2 Channels ◽  
External K

Rat and bovine adrenal zona glomerulosa (ZG) cells possess a low-threshold, voltage-dependent Ca2+ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membrane potentials above -80 mV with maximal peak Ca2+ current elicited near -30 mV. Inactivation of the Ca2+ current was half-maximal between -74 and -58 mV, depending on the external Ca2+ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Cai) levels. Under basal conditions, Cai is stable in single rat ZG cells, whereas more than half of the bovine ZG cells produce repeated Cai transients. These Cai transients, which are blocked by removal of external Ca2+ or addition of Ni2+, are likely due to repetitive electrical activity in bovine ZG cells. Cai responses can be elicited by small increases in external K+ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+ channels. However, these Cai changes remain robust at high external K+ concentrations (20-40 mM). In experiments combining Cai measurements and whole cell voltage clamp, a steep dependence of Cai on membrane potential was revealed beginning at depolarizing voltages near a holding membrane potential of -80 mV. A maximal increase in Cai occurred near -30 mV (equivalent to an external K+ concentration of 40 mM), a membrane voltage at which sustained current through low-threshold Ca2+ channels should be negligible. These data raise the possibility of additional voltage-dependent pathways for Ca2+ influx.

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