Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide

The group-specific protein reagents, N-bromacetamide (NBA) and N- bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur- containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.     

Acid solution is a suitable medium for introducing QX-314 into nociceptors through TRPV1 channels to produce sensory-specific analgesic effects

Background

Previous studies have demonstrated that QX-314, an intracellular sodium channel blocker, can enter into nociceptors through capsaicin-activated TRPV1 or permeation of the membrane by chemical enhancers to produce a sensory-selective blockade. However, the obvious side effects of these combinations limit the application of QX-314. A new strategy for targeting delivery of QX-314 into nociceptors needs further investigation. The aim of this study is to test whether acidic QX-314, when dissolves in acidic solution directly, can enter into nociceptors through acid-activated TRPV1 and block sodium channels from the intracellular side to produce a sensory-specific analgesic effect.

Methodology/Principal Findings

Acidic solution or noradrenaline was injected intraplantarly to induce acute pain behavior in mice. A chronic constrictive injury model was performed to induce chronic neuropathic pain. A sciatic nerve blockade model was used to evaluate the sensory-specific analgesic effects of acidic QX-314. Thermal and mechanical hyperalgesia were measured by using radiant heat and electronic von Frey filaments test. Spinal Fos protein expression was determined by immunohistochemistry. The expression of p-ERK was detected by western blot assay. Whole cell clamp recording was performed to measure action potentials and total sodium current in rats DRG neurons. We found that pH 5.0 PBS solution induced behavioral hyperalgesia accompanied with the increased expression of spinal Fos protein and p-ERK. Pretreatment with pH 5.0 QX-314, and not pH 7.4 QX-314, alleviated pain behavior, inhibited the increased spinal Fos protein and p-ERK expression induced by pH 5.0 PBS or norepinephrine, blocked sodium currents and abolished the production of action potentials evoked by current injection. The above effects were prevented by TRPV1 channel inhibitor SB366791, but not by ASIC channel inhibitor amiloride. Furthermore, acidic QX-314 employed adjacent to the sciatic nerve selectively blocked the sensory but not the motor functions in naïve and CCI mice.

Conclusions/Significance

Acid solution is a suitable medium for introducing QX-314 into nociceptors through TRPV1 channels to produce a sensory-specific analgesic effect.     

Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin.

A voltage clamp technique was used to study sodium currents and gating currents in squid axons internally perfused with the membrane impermeant sodium channel blocker, QX-314. Block by QX-314 is strongly and reversibly enhanced if a train of depolarizing pulses precedes the measurement. The depolarization-induced block is antagonized by external sodium. This antagonism provides evidence that the blocking site for the drug lies inside the channel. Depolarization-induced block of sodium current by QX-314 is accompanied by nearly twofold reduction in gating charge movement. This reduction does not add to a depolarization-induced immobilization of gating charge normally present and believed to be associated with inactivation of sodium channels. Failure to act additively suggests that both, inactivation and QX-314, affect the same component of gating charge movement. Judged from gating current measurement, a drug-blocked channel is an inactivated channel. In the presence of external tetrodotoxin and internal QX-314, gating charge movement is always half its normal size regardless of conditioning, as it QX-314 is then permanently present in the channel.     

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.     

Gsα Protein-Mediated and Protein Kinase A-Independent Regulation of Caveolar Sodium Channels in Rat Cardiomyocytes

In electrophysiological experiments using voltage clamp in the whole-cell configuration and recording of the activity of single channels, extracellular application of 1 mM QX-314 (a derivative of triethyllidocaine) completely and reversibly blocked the sodium conductance through channels in the superficial membranes of rat ventricular cardiomyocytes. After removal of QX-314 from the extracellular medium and in the presence of 22 μg/μl of an inhibitor of protein kinase A (PKA), only the tetrodotoxin-insensitive component of sodium currents was found under conditions of activation of β-adrenoreceptors by isoproterenol (10 μM). In this case, sodium currents activated by isoproterenol were completely inhibited under conditions of intracellular application of monoclonal antibodies against caveolin-3. This observation confirms the hypothesis that an increase in the sodium conductance is, in such cases, mediated by activation of sodium channels belonging to the caveolar pools. The obtained data indicate that subcellular localization of Na_v1.5 channels in caveolar membrane pools of cardiomyocytes can play a special functional role in the enhancement of sodium conductance and modulation of action potentials in rat ventricular cardiomyocytes. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 10–18, January–February, 2009.     

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

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.     

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.     

Sodium inactivation mechanism modulates QX-314 block of sodium channels in squid axons.

Blocking action of Na channels by QX-314, a quaternary derivative of lidocaine, was studied in internally perfused and voltage-clamped axons of squid. In axons with intact Na inactivation, QX-314 exhibited both a frequency- and a voltage-dependent block of Na channels. Repetitive pulsing to more positive potentials enhanced the degree of block. Both frequency- and voltage-dependent blocks disappeared in axons in which Na inactivation had been destroyed by either pronase or N-bromoacetamide treatment. These results support the notion that Na inactivation not only modulates the frequency-dependent block but also involves the voltage-dependent binding reaction between QX-314 and Na channels.     

QX-314 restores gating charge immobilization abolished by chloramine-T treatment in squid giant axons.

The gating status of the QX-314 bound Na channels before and after suppressing the fast inactivation by chloramine-T (CT) was investigated by studying the gating charge immobilization using the OFF gating current (Ig,OFF). CT treatment, which abolishes the charge immobilization induced by a prolonged depolarization, altered the kinetics of Ig,OFF: the fast phase became insensitive to the pulse duration and the slow phase became three times faster than the control one. However, internally applied QX-314 (in the presence of external TTX) caused an immediate charge immobilization similar to that observed in the absence of CT treatment. The Ig,OFF exhibited kinetics similar to the inactivated channels, decaying with a very fast time course. We conclude that the charge immobilization is restored by QX-314 in the chloramine-T-treated axon and that the gating state of the QX-314-bound channel is similar to the inactivated one. The role of the gating charge immobilization in the use-dependent block mechanism is discussed.     

Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons.

The time course of recovery from use-dependent block of sodium channels caused by local anesthetics was studied in squid axons. In the presence of lidocaine or its quaternary derivatives, QX-222 and QX-314, or 9-aminoacridine (9-AA), recovery from use-dependent block occurred in two phases: a fast phase and a slow phase. Only the fast phase was observed in the presence of benzocaine. The fast phase had a time constant of several milliseconds and resembled recovery from the fast Na inactivation in the absence of drug. Depending on the drug present, the magnitude of the time constant of the slow phase varied (for example at -80 mV): lidocaine, 270 ms; QX-222, 4.4 s; QX-314, 17 s; and 9-AA, 14 s. The two phases differed in the voltage dependence of recovery time constants. When the membrane was hyperpolarized, the recovery time constant for the fast phase was decreased, whereas that for the slow phase was increased for QX-compounds and 9-AA or unchanged for lidocaine. The fast phase is interpreted as representing the unblocked channels recovering from the fast Na inactivation, and the slow phase as representing the bound and blocked channels recovering from the use-dependent block accumulated by repetitive depolarizing pulse. The voltage dependence of time constants for the slow recovery is consistent with the m-gate trapping hypothesis. According to this hypothesis, the drug molecule is trapped by the activation gate (the m-gate) of the channel. The cationic form of drug molecule leaves the channel through the hydrophilic pathway, when the channel is open. However, lidocaine, after losing its proton, may leave the closed channel rapidly through the hydrophobic pathway.     

Statistical analysis of single sodium channels. Effects of N-bromoacetamide

Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent.     

Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.     

Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.     

Lighting a backfire to quench the blaze: a combined drug approach targeting the vanilloid receptor TRPV1

Drug interactions and drug specificity are core themes for the pharmacologist. The paper discussed in this Viewpoint exploits the former to attain the latter. How can one improve local anesthetics so that they block pain but permit normal sensation? QX-314 is a charged derivative of lidocaine without anesthetic activity because it cannot diffuse across the cell membrane to access the neuronal voltage-dependent sodium channel. Capsaicin is a selective activator of the TRPV1 channel, the localization of which is restricted to sensory C-fiber neurons involved in nociception. Because the large pore size of the activated TRPV1 allows passage of large cations such as QX-314, combined treatment with capsaicin and QX-314 puts QX-314 uniquely into that subclass of neurons mediating pain, thereby achieving sensational specificity.     

Effects of local anesthetics on properties of tetrodotoxin-sensitive protein reconstituted into liposomes

The effects of some local anesthetics on properties of tetrodotoxin (TTX)-sensitive protein reconstituted into liposomes in such a manner that its TTX-sensitive center is located at the internal surface of the liposome membrane were studied. It was shown that tetracaine, lidocaine and its derivative QX-314 decreased the rate of efflux of radioactive sodium from the²²Na-preloaded proteoliposomes with the same efficiency as TTX acted from the inside of liposomes. The results confirm our earlier suggestion that TTX-sensitive protein is a soluble precursor of the protein forming voltage-dependent sodium channels.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 299–302, July–August, 1995.     

State-dependent block underlies the tissue specificity of lidocaine action on batrachotoxin-activated cardiac sodium channels.

We have identified two kinetically distinct modes of block, by lidocaine, of cardiac sodium channels, activated by batrachotoxin and incorporated into planar lipid bilayers. Here, we analyze the slow blocking mode which appears as a series of nonconducting events that increase in frequency and duration with increasing lidocaine concentrations. This type of block occurred rarely, if at all, for the skeletal muscle sodium channel subtype. Kinetic analysis showed that a linear open-closed-blocked model is sufficient to account for the major features of our data. Slow block occurs from a long closed state that is a distinguishing characteristic of cardiac channels under these conditions. Slow block showed no significant voltage dependence in the range of -60 to -20 mV for which the detailed kinetic analysis was performed, and was not elicited by application of the permanently charged lidocaine derivative QX-314. By contrast, the fast block, described in the companion paper, results from drug binding to the open state, and is similar for cardiac and skeletal muscle sodium channels. Application of trypsin to the cytoplasmic end of the channel eliminates both the spontaneous, long, gating closures and slow block. Thus, the lidocaine-sensitive closed state of batrachotoxin-activated cardiac sodium channels exhibits a protease susceptibility resembling that of the inactivated state of unmodified sodium channels. It is the slow block caused by lidocaine binding to this closed state that underlies the channel-subtype specificity of lidocaine action in our experiments.     

BOLD fMRI of C-Fiber Mediated Nociceptive Processing in Mouse Brain in Response to Thermal Stimulation of the Forepaws

Functional magnetic resonance imaging (fMRI) in rodents enables non-invasive studies of brain function in response to peripheral input or at rest. In this study we describe a thermal stimulation paradigm using infrared laser diodes to apply noxious heat to the forepaw of mice in order to study nociceptive processing. Stimulation at 45 and 46°C led to robust BOLD signal changes in various brain structures including the somatosensory cortices and the thalamus. The BOLD signal amplitude scaled with the temperature applied but not with the area irradiated by the laser beam. To demonstrate the specificity of the paradigm for assessing nociceptive signaling we administered the quaternary lidocaine derivative QX-314 to the forepaws, which due to its positive charge cannot readily cross biological membranes. However, upon activation of TRPV1 channels following the administration of capsaicin the BOLD signal was largely abolished, indicative of a selective block of the C-fiber nociceptors due to QX-314 having entered the cells via the now open TRPV1 channels. This demonstrates that the cerebral BOLD response to thermal noxious paw stimulation is specifically mediated by C-fibers.     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.     

Induction of TRPV1 desensitization by a biased receptor agonist

Selective suppression of hyperactive sensory neurons is an attractive strategy for managing pathological pain. Blocking Na(+) channels to eliminate action potentials and desensitizing transduction channels can both reduce sensory neuron excitability. The novel synthetic vanilloid ligand cap-ET preserves agonist activation of intracellular Ca(2+) signals and large organic cation transport but loses effective electric current induction. Cap-ET can therefore be used to deliver the membrane impermeable Na(+) channel blocker QX-314 to substantially inhibit voltage-activated Na(+) currents. We explored, besides facilitating entry of organic cationic therapeutics, whether cap-ET can also produce receptor desensitization similar to the natural agonist capsaicin. Using the YO-PRO-1 based fluorescent dye uptake assay, we found that cap-ET effectively triggered Ca(2+) dependent desensitization of TRPV1 when the receptor was pre-sensitized with the surrogate oxidative chemical phenylarsine oxide (PAO), suggesting an alternative use of permanently charged cationic capsaicinoids in differential neuronal silencing.