Evidence for a Multivalent Interaction of Symmetrical, N-Linked, Lidocaine Dimers with Voltage-Gated Na+ Channels (original) (raw)
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British Journal of Pharmacology, 2004
Voltage-gated Na þ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Na v 1.7 and Na v 1.8 dorsal root ganglion Na þ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Na v 1.7 and Na v 1.8 Na þ channels were coexpressed with the b 1-subunit in Xenopus oocytes. Na þ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC 50 (dose producing 50% maximum current inhibition) (450 mM for Na v 1.7 and 104 mM for Na v 1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Na v 1.7 being the most sensitive. Only the steady-state activation of Na v 1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Na v 1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Na v 1.7 and Na v 1.8 Na þ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.
British Journal of Pharmacology, 2004
1 The interaction of lidocaine-like local anaesthetics with voltage-operated sodium channels is traditionally assumed to be characterized by tighter binding of the drugs to depolarized channels. As inactivated and drug-bound channels are both unavailable on depolarization, an indirect approach is required to yield estimates for the dissociation constants from channels in inactivated states. The established model, originally described by Bean et al., describes the difference in affinity between resting and inactivated states in terms of the concentration dependence of the voltage shift in the availability curve. We have tested the hypothesis that this model, which assumes a simple Langmuir relationship, could be improved by introducing a Hill-type exponent, which would take into account potential sources of cooperativity.
The Journal of General Physiology, 2004
According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+channels with higher affinities. However, an alternative view suggests that activation of Na+channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n= 5) and 81.7 ± 10.6 μM (n= 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of in...
British Journal of Pharmacology, 2002
1 The structural features that determine the state-dependent interaction of local anaesthetics with voltage-operated sodium channels are still a matter of debate. We have studied the blockade of sodium channels by 2,6-dimethylphenol, a phenol derivative which resembles the aromatic tail of lidocaine, etidocaine, and bupivacaine. 2 The eects of 2,6-dimethylphenol were studied on heterologously (HEK 293) expressed rat neuronal (rat brain IIA) and human skeletal muscle (hSkM1) sodium channels using whole-cell voltage-clamp experiments. 3 2,6-Dimethylphenol was eective in blocking whole-cell sodium inward currents. Its potency was comparable to the potency of lidocaine previously obtained with similar protocols by others. The IC 50 at 770 mV holding potential was 150 and 187 mM for the skeletal muscle and the neuronal isoform, respectively. In both isoforms, the blocking potency increased with the fraction of inactivated channels at depolarized holding potentials. However, the block achieved at 770 mV with respect to 7150 mV holding potential was signi®cantly higher only in the skeletal muscle isoform. The estimated dissociation constant K d from the inactivated state was 25 mM and 28 mM in the skeletal muscle and the neuronal isoform, respectively. The kinetics of drug equilibration between resting and inactivated channel states were about 10 fold faster compared with lidocaine. 4 Our results show that the blockade induced by 2,6-dimethylphenol retains voltage-dependency, a typical feature of lidocaine-like local anaesthetics. This is consistent with the hypothesis that thè aromatic tail' determines the state-dependent interaction of local anaesthetics with the sodium channel.
Anesthesia & Analgesia, 2018
BACKGROUND: Cardiotoxic effects of local anesthetics (LAs) involve inhibition of NaV1.5 voltage-gated Na+ channels. Metastatic breast and colon cancer cells also express NaV1.5, predominantly the neonatal splice variant (nNaV1.5) and their inhibition by LAs reduces invasion and migration. It may be advantageous to target cancer cells while sparing cardiac function through selective blockade of nNaV1.5 and/or by preferentially affecting inactivated NaV1.5, which predominate in cancer cells. We tested the hypotheses that lidocaine and levobupivacaine differentially affect (1) adult (aNaV1.5) and nNaV1.5 and (2) the resting and inactivated states of NaV1.5. METHODS: The whole-cell voltage-clamp technique was used to evaluate the actions of lidocaine and levobupivacaine on recombinant NaV1.5 channels expressed in HEK-293 cells. Cells were transiently transfected with cDNAs encoding either aNaV1.5 or nNaV1.5. Voltage protocols were applied to determine depolarizing potentials that either...
Interaction of local anesthetics with a peptide encompassing the IV/S4–S5 linker of the Na+ channel
Biophysical Chemistry, 2006
The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na + channel, residues 1644-1664. 2 The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1 H-15 N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Δδ( 1 H-15 N), show that benzocaine affects residues L 1653 , M 1655 and S 1656 while lidocaine slightly perturbs residues I 1646 , L 1649 and A 1659 , L 1660 , near the N-and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H 2 O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na + channel functionality as well as the various facets of LA pharmacological activity are proposed in this work. (permanent address).
Journal of Neurophysiology, 2007
Nociceptive neurons of the dorsal root ganglion (DRG) express a combination of rapidly gating TTX-sensitive and slowly gating TTX-resistant Na currents, and the channels that produce these currents have been cloned. The Nav1.7 and Nav1.8 channels encode for the rapidly inactivating TTX-sensitive and slowly inactivating TTX-resistant Na currents, respectively. Although the Nav1.7 channel expresses well in cultured mammalian cell lines, attempts to express the Nav1.8 channel using similar approaches has been met with limited success. The inability to heterologously express Nav1.8 has hampered detailed characterization of the biophysical properties and pharmacology of these channels. In this study, we investigated the determinants of Nav1.8 expression in tsA201 cells, a transformed variant of HEK293 cells, using a combination of biochemistry, immunochemistry, and electrophysiology. Our data indicate that the unusually low expression levels of Nav1.8 in tsA201 cells results from a traff...
The Journal of General Physiology, 2004
According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na ϩ channels with higher affinities. However, an alternative view suggests that activation of Na ϩ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na ϩ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC 50) for the open-channel block at ϩ 30 mV by lidocaine and benzocaine were 20.9 Ϯ 3.3 M (n ϭ 5) and 81.7 Ϯ 10.6 M (n ϭ 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC 50 values for resting-channel block at Ϫ 140 mV were Ͼ 12-fold higher than those for open-channel block. With 300 M benzocaine, rapid time-dependent block (ഠ 0.8 ms) of inactivation-deficient Na ϩ currents occurred at ϩ 30 mV, but such a rapid time-dependent block was not evident at Ϫ 30 mV. The peak current at Ϫ 30 mV, however, was reduced more severely than that at ϩ 30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na ϩ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.
Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons
Neuroscience Research, 2013
Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na + channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (I ACh s) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric ␣34 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and I ACh s were elicited by fast application of ACh (100-300 M), either alone or in presence of lidocaine at different concentrations.