scholarly journals Probing an Open CFTR Pore with Organic Anion Blockers

2002 ◽  
Vol 120 (5) ◽  
pp. 647-662 ◽  
Author(s):  
Zhen Zhou ◽  
Shenghui Hu ◽  
Tzyh-Chang Hwang
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Organic Anion ◽  
Kinetic Studies ◽  
Transmembrane Conductance Regulator ◽  
Fast Kinetics ◽  
Slow Kinetics ◽  
Kd Values ◽  
Voltage Dependent ◽  
Physical And Chemical

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl− current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose–response relationships of both blockers follow a simple Michaelis-Menten function with Kd values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl− increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl− binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl− concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl− and isethionate compete for a binding site in the pore. The voltage dependence and external Cl− concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.

scholarly journals Mg2+ Modulates Voltage-Dependent Activation in Ether-à-Go-Go Potassium Channels by Binding between Transmembrane Segments S2 and S3

2000 ◽  
Vol 116 (5) ◽  
pp. 663-678 ◽  
Author(s):  
William R. Silverman ◽  
Chih-Yung Tang ◽  
Allan F. Mock ◽  
Kyung-Bong Huh ◽  
Diane M. Papazian
Keyword(s):  
Potassium Channels ◽  
Binding Site ◽  
Voltage Sensor ◽  
Wild Type ◽  
Activation Kinetics ◽  
Fast Kinetics ◽  
K Channels ◽  
Transmembrane Segments ◽  
Acidic Residues ◽  
Voltage Dependent

Extracellular Mg2+ directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg2+ presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K+ channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg2+ on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+-sensitivity and mimicked the slowing of activation kinetics caused by Mg2+ binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg2+, indicating that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg2+-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg2+ sensitivity but is in the vicinity of the binding site. We conclude that Mg2+ binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K+ channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.

Using lithium to probe sequential cation interactions with GAT1

2012 ◽  
Vol 302 (11) ◽  
pp. C1661-C1675 ◽  
Author(s):  
Anne-Kristine Meinild ◽  
Ian C. Forster
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Voltage Dependence ◽  
Cation Binding ◽  
Experimental Conditions ◽  
Net Charge ◽  
Cation Binding Site ◽  
Voltage Dependent ◽  
Experimental Findings ◽  
Kinetics Of

Li+ interacts with the Na+/Cl−-dependent GABA transporter, GAT1, under two conditions: in the absence of Na+ it induces a voltage-dependent leak current; in the presence of Na+ and GABA, Li+ stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na+ and Li+ ions at the so-called “ Na2” binding site have been identified, but how Li+ affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and 22Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na+ was partially substituted by Li+. Three novel findings are: 1) Li+ reduced the coupling ratio between Na+ and net charge translocated during GABA cotransport; 2) Li+ increased the apparent Na+ affinity without changing its voltage dependence; 3) Li+ altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na+ or Li+ ion can bind at the putative first cation binding site ( Na2). This is followed by the cooperative binding of the second Na+ ion at the second cation binding site ( Na1) and then binding of GABA. With Li+ bound to Na2, the second Na+ ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.

scholarly journals Multiple open channel states revealed by lidocaine and QX-314 on rat brain voltage-dependent sodium channels.

1996 ◽  
Vol 107 (6) ◽  
pp. 743-754 ◽  
Author(s):  
B C Salazar ◽  
C Castillo ◽  
M E Díaz ◽  
E Recio-Pinto
Keyword(s):  
Binding Site ◽  
Sodium Channels ◽  
Conformational Changes ◽  
Local Anesthetic ◽  
Open Channel ◽  
Amplitude Distribution ◽  
Distribution Analysis ◽  
Kd Values ◽  
Voltage Dependent ◽  
Subconductance States

We have recently reported that brain sodium channels display periods with high (low-Kd) and low (high-Kd) levels of lidocaine-induced open channel block (Salazar, B.C., D.O. Flash, J.L. Walewski, and E. Recio-Pinto. 1995. Brain Res. 699:305-314). In the present study, we further characterize this phenomenon by studying the effects of the permanently charged lidocaine analogue, QX-314. We found that the detection of high- and low-Kd periods does not require the presence of the uncharged form of lidocaine. The level of block, for either period, at various QX-314 concentrations indicated the presence of a single local anesthetic binding site. Increasing the concentration of QX-314 decreased the lifetime of the high-Kd periods while it increased the lifetime of the low-Kd periods. These results could be best fitted to a model with two open channel conformations that display different local anesthetic Kd values (low and high Kd), and in which the channel area defining the local anesthetic Kd consists of multiple interacting regions. Amplitude distribution analysis showed that changes in the Kd values reflected changes in the kon rates, without changes in the koff rates. Both lidocaine and QX-314 were found to be incapable of blocking small-channel subconductance states (5-6 pS). Changes in the local anesthetic kon rates for blocking the fully open state and the lack of local anesthetic block of the small subconductance state are consistent with the presence of channel conformational changes involving the intracellular permeation pathway leading to the local anesthetic binding site.

scholarly journals Stopped-flow fluorescence kinetics of bovine α2-antiplasmin inhibition of bovine midiplasmin

1995 ◽  
Vol 305 (1) ◽  
pp. 97-102 ◽  
Author(s):  
S Christensen ◽  
L Sottrup-Jensen ◽  
U Christensen
Keyword(s):  
Binding Site ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Reaction Model ◽  
Stopped Flow ◽  
Reaction Step ◽  
Fast Kinetics ◽  
Autocatalytic Cleavage ◽  
Series Of Experiments ◽  
The One

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

scholarly journals Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

1993 ◽  
Vol 102 (1) ◽  
pp. 1-23 ◽  
Author(s):  
N A McCarty ◽  
S McDonough ◽  
B N Cohen ◽  
J R Riordan ◽  
N Davidson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Open Channel ◽  
Transmembrane Conductance Regulator ◽  
Channel Block ◽  
Transmembrane Conductance ◽  
Open Channel Block ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Cystic Fibrosis Transmembrane

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

scholarly journals Frequency-dependent amplification of stretch-evoked excitatory input in spinal motoneurons

2012 ◽  
Vol 108 (3) ◽  
pp. 753-759 ◽  
Author(s):  
Randall K. Powers ◽  
Paul Nardelli ◽  
T. C. Cope
Keyword(s):  
Membrane Potential ◽  
Low Frequency ◽  
Excitatory Input ◽  
Medial Gastrocnemius ◽  
Frequency Dependent ◽  
Fast Kinetics ◽  
Synaptic Inputs ◽  
Slow Kinetics ◽  
Voltage Dependent ◽  
Inward Currents

Voltage-dependent calcium and sodium channels mediating persistent inward currents (PICs) amplify the effects of synaptic inputs on the membrane potential and firing rate of motoneurons. CaPIC channels are thought to be relatively slow, whereas the NaPIC channels have fast kinetics. These different characteristics influence how synaptic inputs with different frequency content are amplified; the slow kinetics of Ca channels suggest that they can only contribute to amplification of low frequency inputs (<5 Hz). To characterize frequency-dependent amplification of excitatory postsynaptic potentials (EPSPs), we measured the averaged stretch-evoked EPSPs in cat medial gastrocnemius motoneurons in decerebrate cats at different subthreshold levels of membrane potential. EPSPs were produced by muscle spindle afferents activated by stretching the homonymous and synergist muscles at frequencies of 5–50 Hz. We adjusted the stretch amplitudes at different frequencies to produce approximately the same peak-to-peak EPSP amplitude and quantified the amount of amplification by expressing the EPSP integral at different levels of depolarization as a percentage of that measured with the membrane hyperpolarized. Amplification was observed at all stretch frequencies but generally decreased with increasing stretch frequency. However, in many cells the amount of amplification was greater at 10 Hz than at 5 Hz. Fast amplification was generally reduced or absent when the lidocaine derivative QX-314 was included in the electrode solution, supporting a strong contribution from Na channels. These results suggest that NaPICs can combine with CaPICs to enhance motoneuron responses to modulations of synaptic drive over a physiologically significant range of frequencies.

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 Locale and chemistry of spermine binding in the archetypal inward rectifier Kir2.1

2010 ◽  
Vol 135 (5) ◽  
pp. 495-508 ◽  
Author(s):  
Harley T. Kurata ◽  
Emily A. Zhu ◽  
Colin G. Nichols
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
Before And After ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kinetics Of

Polyamine block of inwardly rectifying potassium (Kir) channels underlies their steep voltage dependence observed in vivo. We have examined the potency, voltage dependence, and kinetics of spermine block in dimeric Kir2.1 constructs containing one nonreactive subunit and one cysteine-substituted subunit before and after modification by methanethiosulfonate (MTS) reagents. At position 169C (between the D172 “rectification controller” and the selectivity filter), modification by either 2-aminoethyl MTS (MTSEA) or 2-(trimethylammonium)ethyl MTS (MTSET) reduced the potency and voltage dependence of spermine block, consistent with this position overlapping the spermine binding site. At position 176C (between D172 and the M2 helix bundle crossing), modification by MTSEA also weakened spermine block. In contrast, MTSET modification of 176C dramatically slowed the kinetics of spermine unblock, with almost no effect on potency or voltage dependence. The data are consistent with MTSET modification of 176C introducing a localized barrier in the inner cavity, resulting in slower spermine entry into and exit from a “deep” binding site (likely between the D172 rectification controller and the selectivity filter), but leaving the spermine binding site mostly unaffected. These findings constrain the location of deep spermine binding that underlies steeply voltage-dependent block, and further suggest important chemical details of high affinity binding of spermine in Kir2.1 channels—the archetypal model of strong inward rectification.

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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