scholarly journals Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

2020 ◽  
Author(s):  
Rizki Tsari Andriani ◽  
Yoshihiro Kubo
Keyword(s):  
Electric Field ◽  
Voltage Clamp ◽  
Fluorescence Intensity ◽  
Voltage Dependence ◽  
Structural Rearrangements ◽  
Close Proximity ◽  
Voltage Dependent ◽  
Gated Channel ◽  
Electrochromic Effect ◽  
Voltage Sensor Domain

ABSTRACTThe gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of the rat P2X2 during ATP- and voltage-dependent gating by voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field here. We also observed slow and voltage-dependent F changes at Ala337, which reflect the structural rearrangements. Furthermore, we identified that the interaction between Ala337 in TM2 and Phe44 in TM1, located in close proximity in the ATP-bound open state, is critical for activation. Taken together, we propose that the voltage dependence of the interaction in the converged electric field underlies the voltage-dependent gating.

scholarly journals Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Rizki Tsari Andriani ◽  
Yoshihiro Kubo
Keyword(s):  
Electric Field ◽  
Voltage Clamp ◽  
Fluorescence Intensity ◽  
Voltage Dependence ◽  
Structural Rearrangements ◽  
Close Proximity ◽  
Voltage Dependent ◽  
Gated Channel ◽  
Electrochromic Effect ◽  
Voltage Sensor Domain

Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.

scholarly journals Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

2010 ◽  
Vol 135 (2) ◽  
pp. 149-167 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Zhe Lu
Keyword(s):  
Electric Field ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Systematic Investigation ◽  
Selectivity Filter ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
Gated Channel

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide–gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller “diameter” and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

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 Kinetic Analysis of Block of Open Sodium Channels by a Peptide Containing the Isoleucine, Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

1998 ◽  
Vol 111 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Galen Eaholtz ◽  
William N. Zagotta ◽  
William A. Catterall
Keyword(s):  
Electric Field ◽  
Sodium Channel ◽  
Kinetic Analysis ◽  
Sodium Channels ◽  
Rate Constant ◽  
Voltage Dependence ◽  
Bimolecular Reaction ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
The Brain

We analyzed the kinetics of interaction between the peptide KIFMK, containing the isoleucine, phen-ylalanine, and methionine (IFM) motif from the inactivation gate, and the brain type IIA sodium channels with a mutation that disrupts inactivation (F1489Q). The on-rate constant was concentration dependent, consistent with a bimolecular reaction with open sodium channels, while the off rates were unaffected by changes in the KIFMK concentration. The apparent Kd was ∼33 μM at 0 mV. The on rates were voltage dependent, supporting the hypothesis that one or both of the charges in KIFMK enter the membrane electric field. The voltage dependence of block was consistent with the equivalent movement of ∼0.6 electronic charges across the membrane. In contrast, the off rates were voltage independent. The results are consistent with the hypothesis that the KIFMK peptide enters the pore of the open sodium channel from the intracellular side and blocks it.

Serotonin and forskolin modulation of a chloride conductance in cultured identified Aplysia neurons

1987 ◽  
Vol 58 (5) ◽  
pp. 922-939 ◽  
Author(s):  
D. P. Lotshaw ◽  
I. B. Levitan
Keyword(s):  
Cell Culture ◽  
Voltage Clamp ◽  
Primary Cell Culture ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Aplysia Neurons ◽  
Voltage Dependent ◽  
Cl Conductance ◽  
Dependent Mechanism

1. The effect of serotonin (5-HT) and forskolin on a hyperpolarization activated Cl- conductance (gCl-) was studied using voltage-clamp techniques in identified Aplysia neurons maintained in primary cell culture. 2. The hyperpolarization-activated conductance induced by intracellular Cl- loading was carried by Cl- as determined by the following criteria: the extrapolated reversal potential of the current closely approximated the reversal potential of a cholinergic Cl- conductance, the current was not affected by extracellular ion substitutions other than Cl-, extracellular thiocyanate ions reversibly inhibited the current and the current exhibited slow voltage-dependent exponential kinetics similar to those described for the hyperpolarization-activated Cl- current in Aplysia neurons in situ. 3. In the identified neurons B1, B2, R15, and R2, 5-HT or forskolin reversibly inhibited gCl-, suggesting that 5-HT acted via an adenosine 3',5'-cyclic monophosphate-dependent mechanism. 4. Serotonergic inhibition resulted from a change in the voltage dependence of Cl- channel gating.

scholarly journals Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

2015 ◽  
Vol 145 (3) ◽  
pp. 185-199 ◽  
Author(s):  
Guoxia Liu ◽  
Sergey I. Zakharov ◽  
Yongneng Yao ◽  
Steven O. Marx ◽  
Arthur Karlin
Keyword(s):  
Bk Channel ◽  
Α Subunit ◽  
Disulfide Bonding ◽  
Close Proximity ◽  
Voltage Dependent ◽  
Gated Channel ◽  
Extracellular Side ◽  
And Shifts ◽  
Voltage Sensing Domain ◽  
Cytoplasmic Side

The large-conductance, voltage- and Ca2+-gated K+ (BK) channel consists of four α subunits, which form a voltage- and Ca2+-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca2+] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K+ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.

scholarly journals The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications

2018 ◽  
Vol 150 (12) ◽  
pp. 1758-1768 ◽  
Author(s):  
Kevin Michalski ◽  
Erik Henze ◽  
Phillip Nguyen ◽  
Patrick Lynch ◽  
Toshimitsu Kawate
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Atp Release ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Pannexin 1 ◽  
Voltage Dependent ◽  
Gated Channel

Pannexins are a family of ATP release channels important for physiological and pathological processes like blood pressure regulation, epilepsy, and neuropathic pain. To study these important channels in vitro, voltage stimulation is the most common and convenient tool, particularly for pannexin 1 (Panx1). However, whether Panx1 is a voltage-gated channel remains controversial. Here, we carefully examine the effect of N-terminal modification on voltage-dependent Panx1 channel activity. Using a whole-cell patch-clamp recording technique, we demonstrate that both human and mouse Panx1, with their nativeN termini, give rise to voltage-dependent currents, but only at membrane potentials larger than +100 mV. This weak voltage-dependent channel activity profoundly increases when a glycine–serine (GS) motif is inserted immediately after the first methionine. Single-channel recordings reveal that the addition of GS increases the channel open probability as well as the number of unitary conductance classes. We also find that insertions of other amino acid(s) at the same position mimics the effect of GS. On the other hand, tagging the N terminus with GFP abolishes voltage-dependent channel activity. Our results suggest that Panx1 is a channel with weak voltage dependence whose activity can be tuned by N-terminal modifications.

scholarly journals External TEA Block of Shaker K+ Channels Is Coupled to the Movement of K+ Ions within the Selectivity Filter

2003 ◽  
Vol 122 (2) ◽  
pp. 239-246 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Electric Field ◽  
Binding Site ◽  
Voltage Dependence ◽  
Internal Surface ◽  
Selectivity Filter ◽  
Dynamic Simulations ◽  
K Channels ◽  
Voltage Dependent ◽  
Electrostatic Calculations ◽  
Average Position

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving ∼37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located ∼13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.

scholarly journals Functional significance of voltage-dependent conductances in Limulus ventral photoreceptors.

1982 ◽  
Vol 79 (2) ◽  
pp. 211-232 ◽  
Author(s):  
P M O'Day ◽  
J E Lisman ◽  
M Goldring
Keyword(s):  
Voltage Clamp ◽  
Functional Significance ◽  
Voltage Dependence ◽  
Outward Current ◽  
Receptor Potential ◽  
Transient Phase ◽  
Plateau Phase ◽  
Initial Transient ◽  
Voltage Dependent

The influence of voltage-dependent conductances on the receptor potential of Limulus ventral photoreceptors was investigated. During prolonged, bright illumination, the receptor potential consists of an initial transient phase followed by a smaller plateau phase. Generally, a spike appears on the rising edge of the transient phase, and often a dip occurs between the transient and plateau. Block of the rapidly inactivating outward current, iA, by 4-aminopyridine eliminates the dip under some conditions. Block of maintained outward current by internal tetraethylammonium increases the height of the plateau phase, but does not eliminate the dip. Block of the voltage-dependent Na+ and Ca2+ current by external Ni2+ eliminates the spike. The voltage-dependent Ca2+ conductance also influences the sensitivity of the photoreceptor to light as indicated by the following evidence: depolarizing voltage-clamp pulses reduce sensitivity to light. This reduction is blocked by removal of external Ca2+ or by block of inward Ca2+ current with Ni2+. The reduction of sensitivity depends on the amplitude of the pulse, reaching a maximum at or approximately +15 mV. The voltage dependence is consistent with the hypothesis that the desensitization results from passive Ca2+ entry through a voltage-dependent conductance.

Analysis of the Bias Dependent Spectral Response of a-SiC:H p-i-n Photodiode

2002 ◽  
Vol 715 ◽  
Author(s):  
P. Louro ◽  
A. Fantoni ◽  
Yu. Vygranenko ◽  
M. Fernandes ◽  
M. Vieira
Keyword(s):  
Bias Voltage ◽  
Voltage Dependence ◽  
Spectral Response ◽  
Surface Recombination ◽  
Electrical Field ◽  
Field Reversal ◽  
Current Voltage ◽  
Voltage Dependent ◽  
And Inversion ◽  
Device Operation

AbstractThe bias voltage dependent spectral response (with and without steady state bias light) and the current voltage dependence has been simulated and compared to experimentally obtained values. Results show that in the heterostructures the bias voltage influences differently the field and the diffusion part of the photocurrent. The interchange between primary and secondary photocurrent (i. e. between generator and load device operation) is explained by the interaction of the field and the diffusion components of the photocurrent. A field reversal that depends on the light bias conditions (wavelength and intensity) explains the photocurrent reversal. The field reversal leads to the collapse of the diode regime (primary photocurrent) launches surface recombination at the p-i and i-n interfaces which is responsible for a double-injection regime (secondary photocurrent). Considerations about conduction band offsets, electrical field profiles and inversion layers will be taken into account to explain the optical and voltage bias dependence of the spectral response.

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