Stereoselectivity of Bupivacaine in Local Anesthetic–sensitive Ion Channels of Peripheral Nerve 

1999 ◽  
Vol 91 (3) ◽  
pp. 786-786 ◽  
Author(s):  
Carla Nau ◽  
Werner Vogel ◽  
Gunter Hempelmann ◽  
Michael E. Bräu
Keyword(s):  
Ion Channels ◽  
Peripheral Nerve ◽  
Binding Site ◽  
Local Anesthetic ◽  
Rate Constants ◽  
Nerve Fibers ◽  
K Channel ◽  
Channel Block ◽  
K Channels ◽  
Ic50 Values

Background The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)-and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel. Methods In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k(-1)) were determined. Results Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/- SEM) for tonic Na+ channel block were 29+/-3 microM and 44+/-3 microM, respectively. IC50 values for flicker K+ channel block were 0.15+/-0.02 microM and 11+/-1 microM, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k(-1) were 0.83+/-0.13x10(6) M(-1) x S(-1) and 0.13+/-0.03 s(-1), respectively, for R(+)-bupivacaine and 1.90+/-0.20x10(6) M(-1) x s(-1) and 8.3+/-1.0 s(-1), respectively, for S(-)-bupivacaine. Conclusions Bupivacaine block of Na+ channels shows no salient stereoselectivity. Block of flicker K+ channels has the highest stereoselectivity ratio of bupivacaine action known so far. This stereoselectivity derives predominantly from a difference in k(-1), suggesting a tight fit between R(+)-bupivacaine and the binding site. The flicker K+ channel may play an important role in yet unknown toxic mechanisms of R(+)-bupivacaine.

Tonic Blocking Action of Meperidine on Na+and K+Channels in Amphibian Peripheral Nerves

2000 ◽  
Vol 92 (1) ◽  
pp. 147-147 ◽  
Author(s):  
Michael E. Bräu ◽  
E. Dietlind Koch ◽  
Werner Vogel ◽  
Gunter Hempelmann
Keyword(s):  
Peripheral Nerve ◽  
Local Anesthetic ◽  
Peripheral Nerves ◽  
Nerve Fibers ◽  
K Channel ◽  
Delayed Rectifier ◽  
Na Channel ◽  
Na Channels ◽  
K Channels ◽  
Local Anesthetic Agent

Background Among opioids, meperidine (pethidine) also shows local anesthetic activity when applied locally to peripheral nerve fibers and has been used for this effect in the clinical setting for regional anesthesia. This study investigated the blocking effects of meperidine on different ion channels in peripheral nerves. Methods Experiments were conducted using the outside-out configuration of the patch-clamp method applied to enzymatically prepared peripheral nerve fibers of Xenopus laevis. Half-maximal inhibiting concentrations were determined for Na+ channels and different K+ channels by nonlinear least-squares fitting of concentration-inhibition curves, assuming a one-to-one reaction. Results Externally applied meperidine reversibly blocked all investigated channels in a concentration-dependent manner, i.e., voltage-activated Na+ channel (half-maximal inhibiting concentration, 164 microM), delayed rectifier K+ channels (half-maximal inhibiting concentration, 194 microM), the calcium-activated K+ channel (half-maximal inhibiting concentration, 161 microM), and the voltage-independent flicker K+ channel (half-maximal inhibiting concentration, 139 microM). Maximal block in high concentrations of meperidine reached 83% for delayed rectifier K+ channels and 100% for all other channels. Meperidine blocks the Na+ channel in the same concentration range as the local anesthetic agent lidocaine (half-maximal inhibiting concentration, 172 microM) but did not compete for the same binding site as evaluated by competition experiments. Low concentrations of meperidine (1 nM to 1 microM) showed no effects on Na+ channels. The blockade of Na+ and delayed rectifier K+ channels could not be antagonized by the addition of naloxone. Conclusions It is concluded that meperidine has a nonselective inhibitory action on Na+ and K+ channels of amphibian peripheral nerve. For tonic Na+ channel block, neither an opioid receptor nor the the local anesthetic agent binding site is the target site for meperidine block.

Blocking Mechanisms of Ketamine and Its Enantiomers in Enzymatically Demyelinated Peripheral Nerve as Revealed by Single-channel Experiments

1997 ◽  
Vol 86 (2) ◽  
pp. 394-404 ◽  
Author(s):  
Michael E. Brau ◽  
Frank Sander ◽  
Werner Vogel ◽  
Gunter Hempelmann
Keyword(s):  
Potassium Channels ◽  
Peripheral Nerve ◽  
Ion Channel ◽  
Single Channel ◽  
Resting Potential ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Na Channel ◽  
Ic50 Values ◽  
Sodium And Potassium

Background Ketamine shows, besides its general anesthetic effect, a local anesthetic-like action that is due to blocking of peripheral nerve sodium currents. In this study, the stereoselectivity of the blocking effects of the ketamine enantiomers S(+) and R(-) was investigated in sodium and potassium channels in peripheral nerve membranes. Methods Ion channel blockade of ketamine was investigated in enzymatically dissociated Xenopus sciatic nerves in multiple-channel and in single-channel outside-out patches. Results Concentration-effect curves for the Na+ peak current revealed half-maximal inhibiting concentrations (IC50) of 347 microM and 291 microM for S(+) and R(-) ketamine, respectively. The potential-dependent K+ current was less sensitive than the Na+ current with IC50 values of 982 microM and 942 microM. The most sensitive ion channel was the flickering background K+ channel, with IC50 values of 168 microM and 146 microM for S(+) and R(-) ketamine. Competition experiments suggest one binding site at the flicker K+ channel, with specific binding affinities for each of the enantiomers. For the Na+ channel, the block was weaker in acidic (pH = 6.6) than in neutral (pH = 7.4) and basic (pH = 8.2) solutions; for the flicker K+ channel, the block was weaker in acidic and stronger in basic solutions. Conclusions Ketamine blockade of sodium and potassium channels in peripheral nerve membranes shows no stereoselectivity except for the flicker K+ channel, which showed a very weak stereoselectivity in favor of the R(-) form. This potential-insensitive flicker K+ channel may contribute to the resting potential. Block of this channel and subsequent depolarization of the resting membrane potential leads, besides to direct Na+ channel block, to inexcitability via Na+ channel inactivation.

scholarly journals Selective Proton-Mediated Transport by Electrogenic K+-Binding Macrocycles

Chemistry ◽  
2020 ◽  
Vol 2 (1) ◽  
pp. 11-21
Author(s):  
Yu-Hao Li ◽  
Shao-Ping Zheng ◽  
Dawei Wang ◽  
Mihail Barboiu
Keyword(s):  
Ion Channels ◽  
Therapeutic Agents ◽  
K Channel ◽  
Transport Activity ◽  
Selective Activation ◽  
K Channels ◽  
High K ◽  
Carbonyl Cyanide ◽  
Self Assembled ◽  
K Transport

Synthetic K+-binding macrocycles have potential as therapeutic agents for diseases associated with KcsA K+ channel dysfunction. We recently discovered that artificial self-assembled n-alkyl-benzoureido-15-crown-5-ether form selective ion-channels for K+ cations, which are highly preferred to Na+ cations. Here, we describe an impressive selective activation of the K+ transport via electrogenic macrocycles, stimulated by the addition of the carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) proton carrier. The transport performances show that both the position of branching or the size of appended alkyl arms favor high transport activity and selectivity SK+/Na+ up to 48.8, one of the best values reported up to now. Our study demonstrates that high K+/Na+ selectivity obtained with natural KcsA K+ channels is achievable using simpler artificial macrocycles displaying constitutional functions.

scholarly journals Halothane Inhibits an Intermediate Conductance Ca2+-activated K+Channel by Acting at the Extracellular Side of the Ionic Pore

2003 ◽  
Vol 99 (6) ◽  
pp. 1340-1345 ◽  
Author(s):  
Mitsuko Hashiguchi-Ikeda ◽  
Tsunehisa Namba ◽  
Takahiro M. Ishii ◽  
Taizo Hisano ◽  
Kazuhiko Fukuda
Keyword(s):  
Ion Channels ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Gamma Aminobutyric Acid ◽  
Time Constants ◽  
K Channels ◽  
Pore Domain ◽  
Inside Out ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Background Actions of volatile anesthetics on ligand-gated ion channels, such as gamma-aminobutyric acid type A receptors, have been studied extensively. However, actions on other types of channels, such as K+ channels, are poorly understood. The authors previously showed that a Ca2+-activated K+ channel, IK, is sensitive to halothane, whereas SK1, another Ca2+-activated K+ channel, is insensitive. To explore how halothane acts on Ca2+-activated K+ channels, chimeras between IK and SK1 were constructed, and halothane sensitivity was analyzed. Methods IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 microm Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations. Results Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK. Conclusions It is suggested that halothane interacts with the extracellular part of the ionic pore of IK. Whether this type of interaction is involved in the mechanism of anesthetic actions on ligand-gated ion channels warrants further investigation.

scholarly journals Inhibition of the Collapse of the Shaker K+ Conductance by Specific Scorpion Toxins

2004 ◽  
Vol 123 (3) ◽  
pp. 265-279 ◽  
Author(s):  
Froylan Gómez-Lagunas ◽  
Cesar V.F. Batista ◽  
Timoteo Olamendi-Portugal ◽  
Martha E. Ramírez-Domínguez ◽  
Lourival D. Possani
Keyword(s):  
Binding Site ◽  
Selectivity Filter ◽  
Channel Block ◽  
Scorpion Toxins ◽  
K Channels ◽  
Outer Vestibule ◽  
Plausible Model ◽  
K Current ◽  
Do So ◽  
External K

The Shaker B K+ conductance (GK) collapses when the channels are closed (deactivated) in Na+ solutions that lack K+ ions. Also, it is known that external TEA (TEAo) impedes the collapse of GK (Gómez-Lagunas, F. 1997. J. Physiol. 499:3–15; Gómez-Lagunas, F. 2001. J. Gen. Physiol. 118:639–648), and that channel block by TEAo and scorpion toxins are two mutually exclusive events (Goldstein, S.A.N., and C. Miller. 1993. Biophys. J. 65:1613–1619). Therefore, we tested the ability of scorpion toxins to inhibit the collapse of GK in 0 K+. We have found that these toxins are not uniform regarding the capacity to protect GK. Those toxins, whose binding to the channels is destabilized by external K+, are also effective inhibitors of the collapse of GK. In addition to K+, other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K+ channels. The inhibition of the drop of GK follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K+ current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect GK in 0 K+ do so by interacting either with the most external K+ binding site of the selectivity filter (suggesting that the K+ occupancy of only that site of the pore may be enough to preserve GK) or with sites capable of binding K+ located in the outer vestibule of the pore, above the selectivity filter.

scholarly journals Mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling

2020 ◽  
Author(s):  
Mátyás C. Földi ◽  
Krisztina Pesti ◽  
Katalin Zboray ◽  
Tamás Hegedűs ◽  
András Málnási-Csizmadia ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Local Anesthetic ◽  
Binding Sites ◽  
Physiological Function ◽  
Therapeutic Potential ◽  
Inhibition Mechanism ◽  
Channel Block ◽  
Drug Candidates ◽  
Unique Mechanism

AbstractSodium channel inhibitor drugs can exert their effect by either blocking, or modulating the channel. The extent of modulation versus channel block is crucial regarding the therapeutic potential of drug candidates. Modulation can be selective for pathological hyperactivity, while channel block affects vital physiological function as much as pathological activity. Previous results indicated that riluzole, a drug with neuroprotective and antiepileptic effects, may have a unique mechanism of action, where modulation is predominant, and channel block is negligible. We studied the effects of riluzole on rNaV1.4 channels expressed in HEK cells. We observed that inhibition by riluzole disappeared and reappeared at a rate that could not be explained by association/dissociation dynamics. In order to verify the mechanism of non-blocking modulation, we synchronized photolabeling with the voltage clamp protocol of patch-clamp experiments. Using this method, we could bind a photoreactive riluzole analog covalently to specific conformations of the channel. Photolabeling was ineffective at resting conformation, but effective at inactivated conformation, as judged from persisting modulated gating after removal of unbound photoactive drug from the solution. Mutation of the key residue of the local anesthetic binding site (F1579A) did not fully prevent ligand binding and inhibition, however, it eliminated most of the modulation caused by ligand binding. Our results indicate that riluzole binds with highest affinity to the local anesthetic binding site, which transmits inhibition by the unique non-blocking modulation mechanism. Our results also suggest the existence of one or more additional binding sites, with lower affinity, and different inhibition mechanism.

scholarly journals The Interaction of Na+ and K+ in Voltage-gated Potassium Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 195-206 ◽  
Author(s):  
Laszlo Kiss ◽  
David Immke ◽  
Joseph LoTurco ◽  
Stephen J. Korn
Keyword(s):  
Binding Site ◽  
K Channel ◽  
Ionic Selectivity ◽  
K Channels ◽  
Voltage Gated ◽  
High Affinity ◽  
Deactivation Kinetics ◽  
Na Currents ◽  
Apparent Ic50 ◽  
K Currents

Voltage-gated potassium (K+) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K+ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K+ channel that is highly selective for K+ over Na+ in physiological solutions, but conducts Na+ in the absence of K+. Na+ and K+ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na+ currents inactivated more rapidly and deactivated more slowly than K+ currents. Currents carried by 160 mM Na+ were inhibited by external K+ with an apparent IC50 <30 μM. K+ also altered both inactivation and deactivation kinetics of Na+ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K+ were inhibited by external Na+, with an apparent IC50 of ∼100 mM. In contrast to the effects of low [K+] on Na+ current kinetics, Na+ did not affect K+ current kinetics, even at concentrations that inhibited K+ currents by 40–50%. These data suggest that Na+ block of K+ currents did not involve displacement of K+ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K+ channels, differential K+ and Na+ conductance, and the concentration dependence of K+ block of Na+ currents and Na+ block of K+ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.

Inhibition of slow-wave repolarization and Ca(2+)-activated K+ channels by quaternary ammonium ions

1993 ◽  
Vol 264 (3) ◽  
pp. C625-C631 ◽  
Author(s):  
A. Carl ◽  
B. W. Frey ◽  
S. M. Ward ◽  
K. M. Sanders ◽  
J. L. Kenyon
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Slow Wave ◽  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Circular Smooth Muscle

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Local anesthetics potently block a potential insensitive potassium channel in myelinated nerve.

1995 ◽  
Vol 105 (4) ◽  
pp. 485-505 ◽  
Author(s):  
M E Bräu ◽  
C Nau ◽  
G Hempelmann ◽  
W Vogel
Keyword(s):  
Local Anesthetics ◽  
Single Channel ◽  
Distribution Coefficients ◽  
Nerve Fibers ◽  
K Channel ◽  
Na Channel ◽  
Myelinated Nerve ◽  
Time Constants ◽  
Channel Inactivation ◽  
Ic50 Values

Effects of some local anesthetics were studied in patch clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers. Micromolar concentrations of external bupivacaine depolarized the excised membrane considerably. The flicker K+ channel was found to be the most sensitive ion channel to local anesthetics in this preparation. Half-maximum inhibiting concentrations (IC50) for extracellular application of bupivacaine, ropivacaine, etidocaine, mepivacaine, lidocaine, and QX-314 were 0.21, 4.2, 8.6, 56, 220, and > 10,000 microM, respectively. The corresponding concentration-effect curves could be fitted under the assumption of a 1:1 reaction. Application from the axoplasmic side resulted in clearly lower potencies with IC50 values of 2.1, 6.6, 16, 300, 1,200, and 1,250 microM, respectively. The log(IC50)-values of the local anesthetics linearly depended on the logarithm of their octanol:buffer distribution coefficients with two regression lines for the piperidine derivatives and the standard amino-amides indicating an inherently higher potency of the cyclic piperidine series. Amide-linked local anesthetics did not impair the amplitude of the single-channel current but prolonged the time of the channel to be in the closed state derived as time constants tau c from closed-time histograms. With etidocaine and lidocaine tau c was 133 and 7.2 ms, and proved to be independent of concentration. With the most potent bupivacaine time constants of wash in and wash out were 1.8 and 5.2 s for 600 nM bupivacaine. After lowering the extracellular pH from 7.4 to 6.6, externally applied bupivacaine showed a reduced potency, whereas at higher pH of 8.2 the block was slightly enhanced. Intracellular pH of 6.4, 7.2, 8.0 had almost no effect on internal bupivacaine block. It is concluded that local anesthetics block the flicker K+ channel by impeding its gating but not its conductance. The slow blocker bupivacaine and the fast blocker lidocaine compete for the same receptor. Lipophilic interactions are of importance for blockade but besides a hydrophobic pathway, there exists also a hydrophilic pathway to the binding site which could only be reached from the cytoplasmic side of the membrane. Under physiological conditions, blockade of the flicker K+ channel which is more sensitive to bupivacaine than the Na+ channel might lead via membrane depolarization and the resulting sodium channel inactivation to a pronounced block of conduction in thin fibers.

Ca(2+)-dependent and Ca(2+)-permeable ion channels in aortic endothelial cells

1992 ◽  
Vol 263 (6) ◽  
pp. H1827-H1838 ◽  
Author(s):  
B. N. Ling ◽  
W. C. O'Neill
Keyword(s):  
Endothelial Cells ◽  
Ion Channels ◽  
K Channel ◽  
Membrane Hyperpolarization ◽  
Aortic Endothelial Cells ◽  
K Channels ◽  
Initial Activation ◽  
Inwardly Rectifying ◽  
Inside Out ◽  
Aortic Endothelial

We investigated whether osmotic stress would activate specific ion channels in bovine aortic endothelial cells (BAECs). In isotonic medium (290 mosmol/kgH2O), cell-attached patch recordings contained both 165-pS K+ channels activated by depolarization and 40-pS K+ channels activated by 200 nM bradykinin. These inwardly rectifying K+ channels were activated by raising “cytoplasmic” Ca2+ in inside-out patches. BAEC exposed to hypotonic bath (220 mosmol/kg) exhibited a 20% decrease in intracellular K+ content within 5 min. Cell-attached patches revealed biphasic K+ channel activation with hypotonic exposure; initial activation of 165- and 40-pS K+ channels (1–3 min) was followed by a delayed but sustained reactivation of both K+ channels (> 5 min). The delayed reactivation phase was dependent on the presence of external Ca2+ and was attenuated by 10 microM gadolinium. A 28-pS nonselective cation channel (NSCC), which conducted inward Ca2+ current, was also detected during hypotonic exposure. This NSCC was stimulated by hyperpolarization and was blocked by 10 microM gadolinium. In BAEC 1) hypotonic exposure activates Ca(2+)-dependent, 165- and 40-pS K+ channels biphasically; 2) the initial phase is independent of external Ca2+, while the delayed phase requires external Ca2+; and 3) Ca(2+)-permeable, 28-pS NSCCs stimulated by membrane hyperpolarization provide a pathway for external Ca2+ influx.

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