scholarly journals Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction.

1977 ◽  
Vol 69 (4) ◽  
pp. 497-515 ◽  
Author(s):  
B Hille
Keyword(s):  
Node Of Ranvier ◽  
Time Dependent ◽  
Na Channels ◽  
Hydrophobic Region ◽  
Inactivation Curve ◽  
Single Receptor ◽  
Na Currents ◽  
Sodium Inactivation ◽  
To Come ◽  
Drug Receptor

The properties of Na channels of the node of Ranvier are altered by neutral, amine, and quaternary local anesthetic compounds. The kinetics of the Na currents are governed by a composite of voltage- and time-dependent gating processes with voltage- and time-dependent block of channels by drug. Conventional measurements of steady-state sodium inactivation by use of 50-ms prepulses show a large negative voltage shift of the inactivation curve with neutral benzocaine and with some ionizable amines like lidocaine and tetracaine, but no shift is seen with quaternary OX-572. However, when the experiment is done with repetitive application of a prepulse-testpulse waveform, a shift with the quaternary cations (applied internally) is seen as well. 1-min hyperpolarizations of lidocaine- or tetracaine-treated fibers restore two to four times as many channels to the conducting pool as 50-ms hyperpolarizations. Raising the external Ca++ concentration also has a strong unblocking effect. These manipulations do not relieve block in fibers treated with internal quaternary drugs. The results are interpreted in terms of a single receptor in Na channels for the different drug types. Lipid-soluble drug forms are thought to come and go from the receptor via a hydrophobic region of the membrane, while charged and less lipid-soluble forms pass via a hydrophilic region (the inner channel mouth). The hydrophilic pathway is open only when the gates of the channel are open. Any drug form in the channel increases the probability of closing the inactivation gate which, in effect, is equivalent to a negative shift of the voltage dependence of inactivation.

scholarly journals Lidocaine, Carbamazepine, and Imipramine Have Partially Overlapping Binding Sites and Additive Inhibitory Effect on Neuronal Na+Channels

2010 ◽  
Vol 113 (1) ◽  
pp. 160-174 ◽  
Author(s):  
Ya-Chin Yang ◽  
Chen-Syuan Huang ◽  
Chung-Chin Kuo
Keyword(s):  
State Dependence ◽  
Inhibitory Effect ◽  
Drug Binding ◽  
Na Channels ◽  
Inactivation Curve ◽  
Functional Studies ◽  
Drug Binding Site ◽  
Na Currents ◽  
Quantitative Analyses ◽  
The One

Background Despite the structural differences, local anesthetics, anticonvulsants, and tricyclic antidepressants exert similar use-dependent actions against voltage-gated Na channels, which may be contributory to pain control. The authors explore whether these drugs could doubly occupy the channel and exert synergic clinical effect. Methods The authors performed electrophysiologic recordings and quantitative analyses in mutant and native neuronal Na channels to investigate molecular interactions between different drugs. Results The authors demonstrate significant interactions between F1764 and W1716, two residues reported for local anesthetic binding, indicating uncertainties to conclude a common drug-binding site by mutation data. Therefore, the authors performed detailed functional studies in native neurons. Quantitative analyses of the inactivation curve shift argue against effective double occupancy of different drugs. For example, the shift of 20.9 +/- 1.3 mV in the simultaneous presence of 10 microm imipramine, 100 microm lidocaine, and 100 microm phenytoin is consistent with the one-site (21.5 mV) rather than the two-site (30.5-33.8 mV) or three-site (42.7 mV) predictions. However, there is a deviation from the recovery courses predicted by one site if lidocaine or imipramine coexists with anticonvulsants. Moreover, gating state dependence of macroscopic-binding rates markedly differs between imipramine and carbamazepine. Conclusions Carbamazepine, lidocaine, and imipramine bind to a common site with the common aromatic motif. External to the aromatic site, there is another weaker and less gating-dependent site for the tertiary amine chain in the latter two drugs. Concomitant clinical use of these drugs, thus, should have at most a simple additive but not a synergistic inhibitory action on Na currents.

scholarly journals Time-Dependent Block and Resurgent Tail Currents Induced by Mouse β4154–167 Peptide in Cardiac Na+ Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 277-289 ◽  
Author(s):  
Ging Kuo Wang ◽  
Thomas Edrich ◽  
Sho-Ya Wang
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Time Dependent ◽  
Purkinje Neurons ◽  
Na Channels ◽  
Wild Type ◽  
Na Currents ◽  
Resurgent Currents ◽  
Lysine Substitution

Resurgent tail Na+ currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse β4 subunit, β4154–167, acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233–244). To explore these phenotypes in vitro, we characterized β4154–167 actions in inactivation-deficient cardiac hNav1.5 Na+ channels expressed in human embryonic kidney 293t cells. Intracellular β4154–167 from 25–250 μM elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents at 50 mV in a concentration-dependent manner. On and off rates for β4154–167 binding were estimated at 10.1 μM−1s−1 and 49.1 s−1, respectively. Upon repolarization, large tail currents emerged with a slight delay at −140 mV, probably as a result of the rapid unblocking of β4154–167. Near the activation threshold (approximately −70 mV), resurgent tail currents were robust and long lasting. Likewise, β4154–167 induces resurgent currents in wild-type hNav1.5 Na+ channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na+ channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na+ channels opened persistently but became resistant to β4154–167 and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na+ channels resistant to β4154–167. Together, our in vitro studies identify a putative S6-binding site for β4154–167 within the inner cavity of hNav1.5 Na+ channels. Such an S6 receptor readily explains (1) why β4154–167 gains access to its receptor as an open-channel blocker, (2), why bound β4154–167 briefly prevents the activation gate from closing by a “foot-in-the-door” mechanism during deactivation, (3) why BTX inhibits β4154–167 binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates β4154–167 binding.

scholarly journals Effects of strychnine on the sodium conductance of the frog node of Ranvier.

1977 ◽  
Vol 69 (6) ◽  
pp. 915-926 ◽  
Author(s):  
B I Shapiro
Keyword(s):  
Sodium Channels ◽  
Node Of Ranvier ◽  
Scorpion Venom ◽  
Time Dependent ◽  
Channel Block ◽  
Approach To Equilibrium ◽  
Sodium Conductance ◽  
Quaternary Derivative ◽  
Tetraethylammonium Ion ◽  
Sodium Inactivation

Strychnine blocks sodium conductance in the frog node of Ranvier. This block was studied by reducing and slowing sodium inactivation with scorpion venom. The block is voltage and time dependent. The more positive the axoplasm the greater the block and the faster the approach to equilibrium. Some evidence is presented suggesting that only open channels can be blocked. The block is reduced by raising external sodium or lithium but not impermeant cations. A quaternary derivative of strychnine was synthesized and found to have the same action only when applied intracellularly. We conclude that strychnine blocks sodium channels by a mechanism analogous to that by which it blocks potassium channels. The potassium channel block had previously been found to be identical to that by tetraethylammonium ion derivatives. In addition, strychnine resembles procaine and its derivatives in both its structure and the mechanism of sodium channel block.

scholarly journals State-dependent Block of Wild-type and Inactivation-deficient Na+ Channels by Flecainide

2003 ◽  
Vol 122 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ging Kuo Wang ◽  
Corinna Russell ◽  
Sho-Ya Wang
Keyword(s):  
Time Constant ◽  
Open Channel ◽  
Inhibitory Concentration ◽  
Antiarrhythmic Agent ◽  
Na Channels ◽  
Fast Inactivation ◽  
Wild Type ◽  
Open Channel Block ◽  
State Dependent ◽  
Na Currents

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM−1s−1 and 12.2 s−1, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels

1992 ◽  
Vol 68 (4) ◽  
pp. 985-1000 ◽  
Author(s):  
H. Sontheimer ◽  
J. A. Black ◽  
B. R. Ransom ◽  
S. G. Waxman
Keyword(s):  
Spinal Cord ◽  
Membrane Potentials ◽  
Resting Potential ◽  
K Channel ◽  
Na Channels ◽  
Patch Clamp Recording ◽  
K Channels ◽  
Na Currents ◽  
Channel Expression

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.

Partition mechanism of adsorption and the absence of displacement phenomena in the zonal analytical chromatography of proteins on bead 2-hydroxy-3-phenoxypropyl-cellulose

1989 ◽  
Vol 54 (9) ◽  
pp. 2375-2385 ◽  
Author(s):  
Peter Gemeiner ◽  
Eva Hrabárová ◽  
Magdaléna Zacharová ◽  
Albert Breier ◽  
Milan J. Beneš
Keyword(s):  
Reaction Time ◽  
Perchloric Acid ◽  
Adsorption Mechanism ◽  
Spherical Shape ◽  
Molar Ratio ◽  
Temperature Reaction ◽  
Reaction Conditions ◽  
Hydrophobic Region ◽  
Bead Cellulose ◽  
To Come

Hydrophobization of bead cellulose is described, carried out by its alkylation with 1,2-epoxy-3-phenoxypropane under the conditions of acid (perchloric acid, borontrifluoride diethyl etherate) and basic (sodium hydroxide) catalysis. Reaction conditions (temperature, reaction time, molar ratio of reactants) have been determined, allowing the hydrophobization of bead cellulose to be carried out to the largest possible extent while maintaining its spherical shape. The nonstoichiometric mechanism suggested for the adsorption of amphiphilic adsorptives on bead 2-hydroxy-3-phenoxypropyl-cellulose (HPP-C) was checked by means of adsorption of six proteins. It was found that the surface of the hydrophobic segment of the adsorbent must be sufficiently large to be able to come in touch with the hydrophobic region of the protein through its multiple residues. In such cases the partitioning of the protein between the hydrophobic segment present as a liquid-like film and the surrounding solution becomes the predominant step of the adsorption. This adsorption mechanism is also reflected in zonal chromatography on bead HPP-C, as no displacement phenomena could be observed in any of the six proteins used. Retention of these proteins has been affected to a decisive extent by the degree of hydrophobization of HPP-C.

Different voltage dependence of transient and persistent Na+ currents is compatible with modal-gating hypothesis for sodium channels

1994 ◽  
Vol 71 (6) ◽  
pp. 2562-2565 ◽  
Author(s):  
A. M. Brown ◽  
P. C. Schwindt ◽  
W. E. Crill
Keyword(s):  
Pyramidal Neurons ◽  
Voltage Dependence ◽  
Sufficient Evidence ◽  
Na Channel ◽  
Na Channels ◽  
Activation Curve ◽  
Neocortical Neurons ◽  
Na Currents ◽  
Flow Through ◽  
The Difference

1. These experiments tested the hypothesis that the differing voltage dependence of the transient (INa) and persistent (INaP) Na+ currents in neocortical neurons results from the state of inactivation of one type of Na+ channel rather than from the existence of different types of Na+ channels. This question was examined in acutely isolated pyramidal neurons from the sensorimotor cortex of rats by using papain to remove inactivation from INa and comparing the resulting activation curve with that of INaP. 2. In control cells, INaP activated at more negative potentials than INa. Inclusion of papain in the recording pipette removed inactivation from INa and caused the INa activation curve to be shifted leftward to the position of the curve for INaP measured in control cells. Papain greatly increased both INa amplitude and the time to reach peak INa during smaller depolarizations, whereas the difference between control and test currents was reduced during large depolarizations. 3. We conclude that differences in the voltage dependence of INa and INaP activation does not provide sufficient evidence that these currents flow through separate sets of Na+ channels. Instead, our results are consistent with the idea that INaP largely arises from a fraction of the transient Na+ channels that intermittently lose their inactivation.

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