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I was doing some reading about anti-epileptic sodium channel blockers, then wondered how many sodium channels are actually conducting (actively passing ions) at any given time, that is, in an "average, awake" brain?
Unfortunately, there is no way to know how many sodium channels are active (or even present) in one person's brain due to the extremely high variability of channel and neuron density in a population and the lack of methodology around quantifying total number of healthy active synapses (1). Diseases like epilepsy can occur due various points of the sodium signaling process going wrong. It could be sodium channels are deformed and not accepting the proper signal, it could a low density of channel proteins, or it could be damage to the neuron themselves (2-4). No one has examined the "normal/healthy range" of sodium channels in the brain. When categorizing sodium channel deficiency scientists will often compare the number of sodium channels in a healthy brain vs. a disease brain using bioluminescence imaging or some other visualization technique. Bioluminescence specially quantifies the number of active sodium channels by binding causing healthy sodium channels light up and then quantifies density by the brightness of the synapse. This is not a direct quantification of the number of proteins, but I can not think of any technique that allows you to count the number of proteins on a cell without damaging the cell. I'm sorry this isn't technically an answer to your question, but hopefully it provides some helpful references to understand molecular brain protein imaging.
This is a difficult question because we are talking about properties of channels-molecules which can have stochastic opening and closing rates. The question is made even more difficult by the fact that there are multiple types of sodium channels. One can begin to quantify this by recording from many many cells in the awake animal using in-vivo electrophysiology by washing in sodium channel blockers and looking at sodium currents. Conversely you can start doing antibody staining for sodium channels in a brain area -- then take acute slices from that area and do single-channel recordings so you have some sense of number* probability of opening.
19.2 Cardiac Muscle and Electrical Activity
Recall that cardiac muscle shares a few characteristics with both skeletal muscle and smooth muscle, but it has some unique properties of its own. Contractions of the heart (heartbeats) are controlled by specialized cardiac muscle cells called pacemaker cells that directly control heart rate. This property is known as autorhythmicity. Neither smooth nor skeletal muscle can do this. Although cardiac muscle cannot be consciously controlled, the pacemaker cells respond to signals from the autonomic nervous system (ANS) to speed up or slow down the heart rate. The pacemaker cells can also respond to various hormones that modulate heart rate to control blood pressure.
There are two major types of cardiac muscle cells: myocardial contractile cells and myocardial conducting cells. The myocardial contractile cells constitute the bulk (99 percent) of the cells in the atria and ventricles. Contractile cells conduct impulses and are responsible for contractions that pump blood through the body. The myocardial conducting cells (1 percent of the cells) are the autorhythmic cells and form the conduction system of the heart. Except for Purkinje cells, they are generally much smaller than the contractile cells and have few of the myofibrils or filaments needed for contraction. Their function is similar in many respects to neurons, although they are specialized muscle cells. Myocardial conduction cells initiate and propagate the action potential (the electrical impulse) that travels throughout the heart muscle and triggers the contractions that propel the blood.
VOLTAGE-GATED ION CHANNELS
Voltage-Gated Sodium Channels
Voltage-gated sodium channels play an essential role in the initiation and propagation of action potentials in neurons (Mantegazza et al. 2010). Neuronal depolarizations by only a few millivolts, which ordinarily result from activation of synaptic glutamate receptors (mainly AMPA receptors, but also N-methyl- d -aspartate [NMDA] receptors), cause sodium channels to open and enable influx of sodium along its electrochemical gradient. These channels then inactivate within milliseconds. The influx of sodium ions during the brief time that sodium channels are open generates the depolarizing component (upstroke) of the action potential. Although nearly all sodium channels inactivate on depolarization, 𢏁% of the sodium current is noninactivating, resulting in a small persistent sodium current (INaP), which is carried by the same channels as the fast transient current. INaP facilitates epileptic burst firing by reducing the threshold for action potential generation, sustained repetitive firing, and augmentation of depolarizing synaptic currents (Stafstrom 2007). Some ASDs, most notably phenytoin, inhibit INaP, an action that is believed to contribute to their efficacy (Mantegazza et al. 2010).
Voltage-gated sodium channels are multimeric protein complexes, composed of a large α-subunit that forms four subunit-like homologous domains (designated I–IV) and one or more smaller β subunits (Meldrum and Rogawski 2007). The ion-conducting pore is contained within the α-subunit, as are the elements of the channel that mediate its fundamental physiological properties, including rapid inactivation. The α subunits alone are able to form a functional sodium channel, but β subunits (㬡–㬤 are known) can modulate the kinetics and trafficking of the channel (Patino and Isom 2010). Of the 10 known α subunits, using the channel and receptor nomenclature of Alexander et al. (2013), Nav1.1, Nav1.2, Nav1.3, and NaV1.6 are the four most abundantly expressed subunits in the brain (Yu and Catterall 2003 Vacher et al. 2008). Mutations in voltage-gated sodium channels have been associated with various genetic forms of epilepsy (Oliva et al. 2012).
ASDs that protect against seizures through an interaction with voltage-gated sodium channels are commonly referred to as “sodium channel blockers” ( Fig. 1 ). They are among the most frequently used drugs in the treatment of both focal and primary generalized tonic𠄼lonic seizures. Such drugs include phenytoin, carbamazepine, lamotrigine, oxcarbazepine (as well as its active metabolite licarbazepine), rufinamide, and lacosamide. ASDs that interact with voltage-gated sodium channels also show a characteristic “use-dependent” blocking action so that they inhibit high-frequency trains of action potentials much more potently than they attenuate individual action potentials or firing at low frequencies. Because they also show a “voltage-dependence” to their blocking action, sodium-channel-blocking ASDs are more potent at inhibiting action potentials superimposed on a depolarized plateau potential as characteristically occurs with seizures. Thus, importantly, sodium-channel-blocking ASDs preferentially inhibit seizure discharges in relation to normal ongoing neural activity. By virtue of their ability to inhibit the action potential invasion of nerve terminals, sodium-channel-blocking ASDs inhibit the release of diverse neurotransmitters, including glutamate whether this is responsible for the therapeutic activity of the drugs remains uncertain (Waldmeier et al. 1995).
Diverse molecular targets for antiseizure drugs (ASDs) at excitatory glutamatergic synapses. Seizure protection can be conferred by effects on voltage-gated sodium channels, M-type voltage-gated potassium channels (Kv7), and voltage-gated calcium channels located in presynaptic terminals. Additional presynaptic targets include the synaptic vesicle protein SV2A and the 㬒δ accessory subunit of voltage-gated calcium channels. These presynaptic targets may act to diminish glutamate release. Postsynaptic targets include ionotropic glutamate receptors of the N-methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) types.
The binding site for sodium on sodium-channel-blocking ASDs is believed to overlap with the binding site of local anesthetics, which is within the pore of the channel and is formed by the S6 segments of domains I, II, and IV. Sodium-channel-blocking ASDs bind with higher affinity to this site when the channel is in the inactivated state, and, when such a drug is bound, the channel is stabilized in the inactivated state (Mantegazza et al. 2010). When neurons are depolarized and firing rapidly, sodium channels spend a greater amount time in the inactivated state and are able to accumulate bound drug so that they become trapped in the inactivated state. This accounts for the use- and voltage-dependent blocking action that they show. Phenytoin, carbamazepine, and lamotrigine are considered 𠇌lassical” sodium-channel-blocking ASDs. Lacosamide is also believed to induce its therapeutic effects by interacting with sodium channels (Rogawski et al. 2015). However, unlike other sodium-channel-blocking ASDs, lacosamide does not inhibit high-frequency repetitive spike firing on the time scale of hundreds of milliseconds. It does, however, inhibit spike firing in long trains of spikes on the time scale of 1𠄲 sec. It has been proposed that the very slow action of lacosamide is caused by an enhancement of a distinct and poorly understood form of inactivation, referred to as “slow inactivation.” An alternative explanation is that lacosamide binds more slowly to fast-inactivated sodium channels than do the other sodium-channel-blocking ASDs. In any case, the unusually slow development of block produced by lacosamide during high-frequency activity could allow lacosamide to better discriminate between seizure-like pathological firing and normal network activity.
T-Type Voltage-Gated Calcium Channels
Low-voltage-activated (T-type) calcium channels play a major role in the intrinsic thalamocortical oscillations that underlie the spike-wave discharges of generalized absence seizures (Avoli et al. 2001 Huguenard 2002 Lambert et al. 2014). There are three T-type Ca 2+ channel isoforms encoded by separate genes, denoted as Cav3.1 (㬑G), Cav3.2 (㬑H), and Cav3.3 (㬑I). All three T-type calcium channel isoforms are expressed in thalamocortical circuits (Talley et al. 1999). Cav3.1 is prominently expressed in thalamic relay neurons in the dorsal thalamus, which plays a key role in absence seizures Cav3.2𠅊nd to a lesser extent Cav3.3𠅊re prominently expressed in thalamic reticular neurons. All three T-type calcium channel isoforms are expressed in the cortex, with Cav3.2 mainly localized to layer V. In non-REM sleep, including when δ waves, sleep spindles, and K-complexes occur, the thalamocortical circuit switches from a tonic to oscillatory mode of firing, but in absence epilepsy, this switching can occur inappropriately, even during wakefulness (Crunelli et al. 2014 Powell et al. 2014). T-type calcium channels in the thalamus and cortex contribute to the abnormal behavior of the circuit. These channels generate low-threshold spikes, leading to burst firing and oscillatory behavior (Suzuki and Rogawski 1989). GABAergic neurons of the thalamic reticular nucleus are also critically involved in absence seizures as they hyperpolarize thalamic relay neurons, which de-inactivate T-type calcium channels, allowing the channels to generate burst firing and the propagation of spike-wave discharges in the thalamocortical circuit (Danober et al. 1998).
Ethosuximide, which is highly efficacious in the treatment of absence seizures𠅋ut not other seizure types—is thought to act by inhibition of T-type calcium channels in the thalamocortical circuit (Coulter et al. 1989 Broicher et al. 2007 Gören and Onat 2007). At clinically relevant concentrations (20 µg/ml), some but not all investigators have observed a partial (20%%) reduction of T-type calcium current by ethosuximide. Notwithstanding this discrepancy, studies with recombinant T-type calcium channels have confirmed that ethosuximide blocks all three channel types (Gomora et al. 2001). The block increases when the current is activated from more depolarized potentials and when T-type calcium channels are inactivated, as occurs especially during high-frequency activation, so that the drug has selectivity for pathological behavior in the thalamocortical circuit, which is associated with neuronal depolarization and inactivation of T-type calcium channels. Effects on other membrane currents, including INaP, calcium-activated potassium current (Broicher et al. 2007), and inward rectifier potassium current (Huang and Kuo 2015), may contribute to the efficacy of ethosuximide in absence epilepsy. Remarkably, results in animal models indicate that early treatment with ethosuximide can have disease-modifying (i.e., antiepileptogenic) effects, causing a persistent reduction in seizures and mitigation of behavioral comorbidities (Blumenfeld et al. 2008 Dezsi et al. 2013). These actions may be caused by epigenetic modifications. A study showing that children with absence epilepsy who receive ethosuximide are more likely than those who receive valproate to achieve long-term remission is consistent with the disease-modifying actions observed in animal studies (Berg et al. 2014).
The efficacy of some other ASDs may also depend, at least in part, on actions at T-type calcium channels. Zonisamide, in addition to effects on voltage-gated sodium channels, may also block T-type voltage-gated calcium channels (Powell et al. 2014), thus accounting for its likely efficacy in absence epilepsy (Hughes 2009). Similarly, there is evidence that valproate, a drug of choice in absence epilepsy, may also inhibit T-type calcium channels (Broicher et al. 2007).
Kv7 Voltage-Gated Potassium Channels
Voltage-gated potassium channels open in response membrane depolarization, permitting efflux of potassium ions, thus driving the membrane potential toward a hyperpolarized level. This serves to repolarize depolarizing events (such as action potentials and synaptic potentials) and cause a generalized reduction in excitability. In 1998, the first genes for a human idiopathic epilepsy were identified (Charlier et al. 1998). These genes, designated KCNQ2 and KCNQ3, encoded novel brain potassium channel subunits, Kv7.2 and Kv7.3, respectively, which are homologous to a previously identified cardiac potassium channel Kv7.1, encoded by KCNQ1 (LQT1). These brain potassium channels mediate the M current, a potassium current that increases as the membrane potential in neurons approaches action potential threshold. Kv7 channels, together with hyperpolarization-activated cyclic nucleotide-gated potassium (HCN) channels and small-conductance calcium-activated potassium (KCa/SK) channels, generate the medium after-hyperpolarization, which is elicited by a burst of action potentials and serves to limit further firing (Gu et al. 2005). Kv7 potassium channels also serve to counteract the spike after-depolarization generated by recruitment of INaP, which can lead to bursting (Yue and Yaari 2006). Kv7 potassium channels, therefore, act as a 𠇋rake” on epileptic burst firing. The Kv7 family of potassium channels is now known to contain five members, including Kv7.1, which is expressed predominantly in the heart and Kv7.2–Kv7.5, which are expressed exclusively in the nervous system (Brown and Passmore 2009). Of these Kv7 family members, Kv7.2 and Kv7.3 are highly expressed in neurons relevant to epilepsy, including principal (pyramidal) cells of the hippocampus and neocortex. Kv7.2 and Kv7.3 compose heterotetrameric channels in which four subunits are arranged around a potassium selective pore. Kv7.5 channels may also contribute to M current and to neuronal after-hyperpolarization, for example, in the CA3 area of the hippocampus (Tzingounis et al. 2010).
Studies of the localization of Kv7.2 and Kv7.3 potassium channels by immunohistochemical techniques have indicated that the channels are present at highest density in axons and their terminals (Vacher et al. 2008). In myelinated fibers, they are present at nodes of Ranvier and the channels are also expressed at axon initial segments. In addition, the channels are expressed at lower levels in the somata of principal neurons and in some GABAergic neurons. Physiological studies in CA1 pyramidal neurons indicate that Kv7 channels are functionally active in the perisomatic region (Hu et al. 2007) and possibly also on distal dendrites (Yue and Yaari 2006).
Ezogabine (retigabine), which is efficacious in the treatment of focal seizures, acts as a positive modulator of the nervous system Kv7 potassium channels (Kv7.2–Kv7.5), but does not affect the cardiac member of the family (Kv7.1) ( Fig. 1 ). Of particular relevance to the antiseizure action of ezogabine is its action on the M current, which is predominantly carried by channels composed of Kv7.2 and Kv7.3, although Kv7.5 alone or in combination with Kv7.3 also contributes (Rogawski 2006 Gunthorpe et al. 2012). Ezogabine causes a hyperpolarizing shift in the activation of Kv7 channels such that more M current is generated near the resting potential. It also causes a change in the kinetics of single KCNQ channels to favor channel opening, thus increasing the macroscopic M current ezogabine, nevertheless, does not alter the single channel conductance of individual Kv7 channels (Tatulian and Brown 2003). As noted, many Kv7 channels in the brain are believed to be Kv7.2/Kv7.3 heteromers, and these are highly sensitive to ezogabine (EC50, 1.6 µ m ) (Gunthorpe et al. 2012). Peak plasma levels of ezogabine range from 354 to 717 ng/ml (1.2𠄲.4 µ m ) (Hermann et al. 2003), and plasma protein binding is 80% so that free plasma concentrations are estimated to be 𢏀.2𠄰.5 µ m brain concentrations are expected to be similar. Therefore, therapeutic concentrations likely only modestly potentiate the most sensitive Kv7 channels and do not affect less sensitive channels. The binding site for ezogabine in Kv7.2/Kv7.3 heteromers is in a pocket formed by the pore-lining S5 membrane segment of one subunit and the pore-lining S6 membrane segment of the neighboring subunit (Wuttke et al. 2005 Lange et al. 2009). Channel opening may expose the pocket, permitting binding of ezogabine, which stabilizes the open channel conformation.
Several experimental approaches support the role of Kv7 potassium channels in the antiseizure activity of ezogabine. Mice with a genetic defect in these channels show reduced sensitivity to the antiseizure effect of ezogabine (Gunthorpe et al. 2012). Furthermore, the KCNQ inhibitor, XE-991, partially blocks the antiseizure effect of ezogabine in an electrical seizure test (Gunthorpe et al. 2012). However, the precise way in which activation of Kv7 channels leads to seizure protection remains to be elucidated. Consistent with the presynaptic localization of many Kv7 channels, ezogabine has been found to inhibit the release of various neurotransmitters, including GABA and probably also glutamate (Martire et al. 2004). Inhibition of glutamate release is expected to confer seizure protection. The impairment of GABA release or the direct inhibition of inhibitory interneurons (Lawrence et al. 2006 Grigorov et al. 2014) would be expected to enhance circuit excitability. Indeed, in rodents, retigabine shows proconvulsant activity at doses (.9 mg/kg) that are approximately 10-fold greater than those associated with seizure protection (U.S. Food and Drug Administration 2010). Paradoxically, and in contrast to biochemical observations, physiological studies have found that ezogabine “increases” glutamate-mediated synaptic transmission in the hippocampus through a presynaptic action by reducing the inactivation of voltage-gated sodium channels so that sodium-dependent action potentials are elicited with greater probability (Vervaeke et al. 2006). This action could contribute to the proconvulsant effects of the drug. If ezogabine does confer seizure protection through effects on excitatory neurons, postsynaptic actions to inhibit somatic excitability likely predominate.
In addition to its effects on Kv7 potassium channels, ezogabine has been reported to interact with the GABA system. At high concentrations (㸐 µM), ezogabine was shown to potentiate GABA-mediated inhibitory transmission by acting as a positive allosteric modulator of GABAA receptors (Rundfeldt and Netzer 2000 Otto et al. 2002). Recent evidence that inhibitory effects of the drug on seizure-like activity in hippocampal neurons persist in the presence of blockade of Kv7 channels has bolstered the view that positive modulation of GABAA receptors could be key to its antiseizure activity (Treven et al. 2015). Thus, at lower concentrations than are required for effects on synaptic GABAA receptors, ezogabine selectively enhances extrasynaptic GABAA receptors that contain the δ-subunit (Treven et al. 2015). Ezogabine has also been reported to increase GABA synthesis (Kapetanovic et al. 1995) the mechanism and implications of this effect are not known. In sum, various lines of evidence raise the possibility that effects of ezogabine on GABA mechanisms could be of importance in its antiseizure activity.
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Building the homology model
Previously, LAs and other ligands were docked in homology models, which were built using as templates the x-ray structures of KcsA (Lipkind and Fozzard, 2000 Bruhova et al., 2008), MthK (Lipkind and Fozzard, 2005), KvAP (Tikhonov et al., 2006), and NavAb (Tikhonov and Zhorov, 2012). Sequences of eukaryotic sodium channels are obviously much more similar to prokaryotic sodium channels than to potassium channels, and their alignment is much less ambiguous (Table S1). The sequences of bacterial sodium channels with available structures are rather similar, and their comparison does not allow us to select a preferable template. Among these structures, the NavMs bacterial channel (Naylor et al., 2016) presents a template that has three advantages for modeling eukaryotic channels with the inner-pore–bound ligands. First, this sodium channel is crystallized in the apparently open state. Second, this structure shows sodium ions in the selectivity-filter region. Third, the NavMs channel is also cocrystallized with a LA-like ligand (Bagnéris et al., 2014). Thus, using the NavMs template seems promising to advance our understanding of ligand interactions with eukaryotic sodium channels.
We have built the Nav1.4 channel model using the NavMs channel (PDB accession number 5BZB) as the template. The selectivity filter, which is different in bacterial and eukaryotic sodium channels channel, was modeled as in our previous studies (Tikhonov and Zhorov, 2012). A sodium ion NaIII was added to the model near the entrance to the inner pore as seen in the NavMs structure (Naylor et al., 2016). Position of NaIII is also close to site 4 for potassium ions in potassium channels. Other ions, which are seen in the x-ray structure (Naylor et al., 2016), were not added because according to our recent calculations, their electrostatic interactions with the inner-pore ligands are weak (Korkosh et al., 2016). Furthermore, the number of ions in the DEKA ring of eukaryotic channels may differ from that in the EEEE ring of NavMs. The sequence alignment, ligand-sensing residues found by mutational analysis and their positions in the NavMs structure are given in Fig. 1 (B and C). The model was optimized by a two-stage MCM protocol. At the first stage, the backbone torsions were kept rigid. At the second stage, both side-chain and backbone angles were varied and similarity between the template and the model was maintained by “pin” constraints imposed to α carbons (see Materials and methods). According to the MolProbity server (Chen et al., 2010), the quality (overall score) of the model (1.06) is similar to that of the NavMs template (1.27). The only region, where the backbone in our model differs from that of the template is short loops between P1 and P2 helices, in which insertions/deletions were introduced to accommodate experimental data on binding of tetrodotoxin (Tikhonov and Zhorov, 2012).
Hands-free docking of lidocaine and CMZ in the models with and without NaIII
For the initial hands-free docking we selected a protonated LA (lidocaine) and a neutral anticonvulsant (CMZ). Theoretical proposals and experimental data suggest significant interactions between blocking molecules and current-carrying ions (Zhorov and Tikhonov, 2013). Therefore, we docked each ligand in two channel models: with and without a sodium ion at site NaIII (Fig. 2). In agreement with previous studies (Sunami et al., 1997 Tikhonov and Zhorov, 2007), we observed repulsion between the charged lidocaine and the sodium ion NaIII. Positions of the lidocaine amino group in different binding modes were scattered over a wide region in the inner pore (Fig. 2 A). Contrary, in the absence of NaIII, the binding modes were found with the amino group clustered at the cation-attractive region in the pore, including site NaIII (Fig. 2 B).
The hands-free docking of CMZ yielded opposite results. In the model without NaIII, CMZ binding modes were scattered in the pore and interfaces between helices (Fig. 2 C). This result is reminiscent to molecular dynamics (MD) simulations of the NavAb sodium channel with benzocaine and phenytoin, where many possible positions of the ligands were found (Boiteux et al., 2014). In the model with NaIII, low-energy binding modes of CMZ are clustered in the inner pore with the CMZ carbonyl oxygen often bound to NaIII (Fig. 2 D). These calculations suggest that the channel with NaIII favors binding of neutral ligands, whereas the channel without NaIII favors binding of charged ligands. Thus, the NaIII site appears to be the hotspot for binding of both charged and neutral ligands.
Docking lidocaine in the channel model without NaIII
We systematically explored possible binding modes of lidocaine in the model without NaIII (see Materials and methods). These computations yielded an ensemble of 63 low-energy ligand–channel complexes. Let us define axis Z, which coincides with the pore axis, originates at NIII and directs extracellularly. We characterized each binding mode of lidocaine by the Z-coordinate of the ammonium nitrogen, ZN+, its deviation from the pore axis, DN+, and respective coordinates of the aromatic carbon Cp in the para position to the amide nitrogen, ZCp and DCp. Characteristics of the ensemble are given in Fig. 3. Several pore-domain regions accessible for ligands and ions can be defined (Fig. 1 C). In the selectivity filter region (1), −2 Å < Z <5 Å and deviations from the pore axis are <2 Å. The inner-pore region (2) occupies the wide space between the P-loop turns and positions i21 with −10 Å < Z < −2 Å and deviations from the pore axis up to 8 Å. We define the activation gate region (3) as that where S6 helices differ significantly between the closed sodium channel NavAb and apparently open channel NavMs. This region is located below the pore region and has the coordinates Z < −10 Å, and deviations from the pore axis are <5 Å. The four interface regions (4) correspond to four fenestrations between the inner helices. Their main feature is large (>8 Å) deviations from the pore axis.
Distribution of ZN+ has peaks at level i15 (the inner-pore region) and immediately below site NaIII (Fig. 3 A). The ammonium group can approach the pore axis, or S6s, and also can be found in the interfaces or even at the pore domain outer surface (Fig. 3 B). Atom Cp occurs rarely at the NaIII level, most frequently in the pore, and also at the activation gate (Fig. 3 C), where N + is practically not seen (Fig. 3 A). The aromatic ring of lidocaine can bind in the pore or in repeat interfaces (Fig. 3 D).
We further plotted four correlation fields and circled clusters with characteristic locations of N + or/and Cp. In Fig. 3 E, points close to or far from the dashed line (ZCp = ZN+) represent, respectively, approximately horizontal or vertical lidocaine orientations. Among vertical orientations, the cluster dominates where N + and Cp are, respectively, at site NaIII and in the pore. In the second cluster of vertical orientations, N + and Cp are, respectively, in the pore and at the activation gate. In Fig. 3 F, the leftmost cluster represents binding modes with Cp at the activation gate, close to the pore axis because of rather narrow gate aperture. In the largest cluster, Cp is in the inner pore. In the topmost cluster, Cp is in interfaces and may significantly deviate from the pore axis. The lower, middle, and upper clusters in Fig. 3 G represent, respectively, structures where N + is close to site NaIII, approaches S6s from inside the pore, and occurs in interfaces. In Fig. 3 H, binding modes are clustered as in Fig. 3 F. The leftmost cluster represents lidocaine-binding modes where location of Cp at the gate dictates vertical orientation of the ligand with small deviations of N + from the pore axis. In the largest cluster, ZCp varies significantly, whereas N + is close to the pore axis because of restriction imposed by S6s. In the topmost cluster, both N + and Cp are in interfaces. The interface and inner-pore clusters are separated because the ammonium group avoids the narrowest hydrophobic part of the fenestrations such separation is not seen in the ZCp/DCp correlation field (Fig. 3 F).
This analysis revealed three distinct binding modes with similar characteristics within each one. Two of these binding models (Fig. 4, B and D) correspond to previous results where lidocaine is seen in horizontal and vertical binding orientations (Tikhonov and Zhorov, 2007 Bruhova et al., 2008). In one of the vertical binding modes (Fig. 4 D), the ammonium group occurred at the cation-attractive region at the focus of P1 helices. This is a putative position NaIV, which is analogous to site 5 where a potassium ion is seen in the KcsA x-ray structure (Zhou et al., 2001). The ammonium group also approached F 4i15 to form cation-pi contacts. The aromatic group of lidocaine occurred at the activation-gate level and contacted Y 4i22 and I 3i23 , the experimentally known LA-sensing residues (Yarov-Yarovoy et al., 2002). In the horizontal binding mode, the ammonium group is approximately at the same level, but the aromatic moiety extends to repeat interfaces (Fig. 4 B). Different positions of lidocaine in the horizontal mode likely reflect its hydrophobic access route through the sidewalk between repeats III and IV.
A novel finding is the diversity of lidocaine vertical binding modes, which was not revealed in the sodium channel models based on potassium channel templates. Besides the aforementioned binding mode (Fig. 4 D), we found a new mode in which the cationic group approached the selectivity filter (Fig. 4 C) and occurred at the level where NaIII is seen in the NavMs structure (ZN+ is close to zero). The ammonium group formed favorable contacts with the backbone carbonyls in positions p48 and with side chains in position p49. In this mode, the aromatic moiety of lidocaine contacted F 4i15 and Y 4i22 . The aromatic rings of the ligand and F 4i15 formed face-to-face or edge-to-face contacts. Such a binding mode is not proposed in previous models, which are based on potassium channel x-ray structures, because the ligand ammonium group would not fit in the narrow level corresponding to site 4 in potassium channels.
Docking CMZ in the channel model with NaIII
Intensive docking of CMZ in the channel with a sodium ion predicted the ensemble of 80 diverse low-energy structures. In these computations, the sodium ion was initially placed at site NaIII, but its position was randomized. We characterized mutual disposition of CMZ and Na + by the Z-coordinates of the CMZ carbonyl oxygen (ZO) and Na + (ZNa), deviations of these atoms from the pore axis (DO and DNa), and the distance between the CMZ oxygen and Na + , RO,Na+. The CMZ oxygen is seen in the inner pore and immediately below site NaIII (Fig. 5 A). It occurs most frequently in the pore but is also found in interfaces (Fig. 5 B). Na + occurs frequently at site NaIII and is also found inside the pore, at the level corresponding to putative site NaIV or site 5 in potassium channels (Fig. 5 C). The sodium ion most frequently approached the pore axis but can also deviate from it (Fig. 5 D). The RO,Na+ distance distribution (Fig. 5 E) shows that in the vast majority of the structures, Na + is coordinated by the CMZ oxygen. In the correlation field ZNa/DO (Fig. 5 F), the rightmost clusters represent structures with Na + at site NaIII, either bound to or unbound from CMZ. The left cluster combines structures where the CMZ-bound Na + is inside the pore. Correlation field ZNa/DNa (Fig. 5 G) shows groups with Na + at site NaIII or inside the pore. Correlation field ZNa/RO,Na+ (Fig. 5 H) has two clusters. In the largest one, Na + and CMZ are coordinated, but Na + is found either at site NaIII or below it. In the smaller cluster, Na + at site NaIII is not bound to the CMZ oxygen. In the modes where CMZ did not directly interact with NaIII, its aromatic groups interacted with different pore-facing S6 residues, including F 4i15 (Fig. 4 E). When the CMZ-bound Na + was at site NaIII, the CMZ tricyclic moiety contacted F 4i15 (Fig. 4 F) When the CMZ-bound Na + was between levels NaIII and NaIV, the CMZ amino group contacted F 4i15 , whereas its tricyclic moiety reached the activation gate level and contacted residues at positions i22 and i23 (Fig. 4 G).
Similarity of lidocaine and sodium-bound CMZ binding modes
Comparison of low-energy binding modes of lidocaine and sodium-bound CMZ shows their intriguing similarity. First, the amino group of lidocaine and CMZ-bound sodium ion most frequently occurred in very similar positions, between the levels of NaIII and NaIV. Second, the aromatic moieties of both ligands interacted with the pore-facing S6 residues in similar ways. This similarity is caused by the fact that mutual disposition of aromatic and cationic moieties in lidocaine matches that in sodium-bound CMZ.
This finding allowed us to formulate the following working hypothesis. Both charged and neutral ligands bind to the channel in similar ways. The sodium ion bound to a neutral ligand occurs in the same position as the cationic group in a charged ligand and plays an analogous role by electrostatically preventing permeation. In later sections, we elaborate this hypothesis by considering representative examples of charged and neutral ligands that, according to experimental data, target the inner pore of sodium channels. We docked these ligands, obtained ensembles of low-energy structures, and subdivided the ensembles into binding modes with similar characteristics of ligand–channel interactions. Below, we describe these binding modes.
We did not consider multiple LAs that are structurally similar to lidocaine because we did not expect that their docking would produce principally new results. We have chosen QX-314, which blocks the inner pore (Qu et al., 1995), but it was unclear if its triethylammonium group would fit the rather narrow pore at the level of the NaIII site. A similar motivation underlined our choice of even bulkier cocaine and quinidine, for which mutational data suggest binding in the inner pore (Ragsdale et al., 1996 O’Leary and Chahine, 2002). We have also chosen sipatrigine (Liu et al., 2003), an unusually long and rigid derivative of the well-studied anticonvulsant lamotrigine.
In our model, QX-314 bound to the same site and in the same binding mode as lidocaine did. The triethylammonium group fits the NaIII site (Fig. 6 A) without forming repulsive contacts. The positive charge of QX-314, which is distributed over alkyl groups, is electrostatically attracted to the backbone carbonyls in position p48. Besides well-known LA-sensing residues in helices S6, our model predicted that the bulky ammonium group can also contact sidechains in positions p49. Mutations Q 1p49 C and F 3p49 C have been demonstrated to decrease action of QX-314 (Yamagishi et al., 2009). The authors of this study suggested an allosteric mechanism but did not rule out a possibility of direct interaction of residues p49 with QX-314. Our model is consistent with the latter possibility. Furthermore, F 3p49 was predicted to interact with the pore-bound batrachotoxin (Tikhonov and Zhorov, 2005), and intensive mutational study confirmed this prediction (Wang et al., 2006).
Cocaine and quinidine
Unlike flexible alkylammonium groups of lidocaine and QX-313, the ionizable nitrogen in cocaine and quinidine is incorporated into bulky rigid bicyclic moieties. These structural peculiarities did not disfavor binding modes with the ammonium nitrogen at site NaIII (Fig. 6, B–D). Quinidine has a hydroxyl group, which may interact with the polar groups in positions p48 and p49 (Fig. 6 B). The aromatic groups of these ligands bound between F 4i15 and Y 4i22 . For both ligands, the binding modes were also possible with the ammonium group at level i15 (Fig. 6 D).
Because of structural peculiarities of sipatrigine, its contacts with the channel markedly differ from those predicted for other cationic ligands. In the vertical binding mode, sipatrigine extended form the level of NaIII (where its ammonium group bound) to the level of i23 in the activation-gate region (Fig. 6 F). The long and rigid sipatrigine molecule stretched parallel to the pore axis and did not bind tightly against IVS6, as other cationic drugs typically bind. In the horizontal binding mode, sipatrigine stretched from the inner pore deeply into the interface between helices IIIP, IIIS6, and IVS6 (Fig. 6 E). Unlike other cationic ligands, the long sipatrigine molecule established multiple simultaneous contacts with residues both in the inner pore and in the interface.
Among many ligands, we have chosen those that have essentially different chemical structures (Fig. 1 A). Phenytoin has two amide groups in the five-membered ring. Lamotrigine has a triazine ring with two attached amino groups and lacks oxygen atoms. CMZ, which binds in the common site with lamotrigine and phenytoin, has an amide group attached to the bulky tricyclic moiety. Unlike the three semi-rigid ligands, lacosamide is a very flexible compound. We also considered bisphenol A, a pollutant that has two distant hydroxyls and, unlike other compounds, lacks nitrogen atoms. Experimental data strongly suggest binding of these ligands in the inner pore (Ragsdale et al., 1996 Kuo, 1998 Liu et al., 2003 O’Reilly et al., 2012 Wang and Wang, 2014). Despite the great structural diversity, docking of all these compounds revealed binding modes reminiscent to those of CMZ. First, all these compounds interacted with a sodium ion at site NaIII. Second, all these compounds donated H bonds to backbone carbonyls in position p48, thus substituting water molecules, which bridge NaIII to the backbone carbonyls in the ligand-free NavMs (Naylor et al., 2016). Third, nonpolar moieties of the drugs extended into the inner pore between F 4i15 and Y 4i22 in a manner similar to those found for the charged ligands.
In the predicted complexes, the sodium ion was bound to the triazine-ring plane. The amino groups donated H bonds to the backbone carbonyls p48 (Fig. 6, G and H). The dichloro-substituted aromatic ring bound between F 4i15 and Y 4i22 and also contacted V 4i18 . Alanine substitutions of these residues are known to affect action of lamotrigine (Liu et al., 2003).
In the predicted low-energy complexes, the sodium ion was bound to the ligand’s carbonyl oxygen, which is located between two NH groups, and approached an aromatic ring. In this binding mode, the NH groups can donate H bonds to the backbone oxygen atoms in positions p48 and p49. For example, in the structure shown in Fig. 6 L, one NH group donated an H bond to 3p48 O=C and another NH group donated an H bond to the sidechain of Q 1p49 .
Batrachotoxin-activated Nav1.5 channels and the Nav1.5 mutant F 4i15 K are completely resistant to the lacosamide block, indicating that the lacosamide receptor overlaps with those for batrachotoxin and LAs (Wang and Wang, 2014). Only (R)-isomer of lacosamide is active (Porter et al., 2012). A very flexible lacosamide has three oxygen atoms and an aromatic ring capable of interacting with Na + . Therefore, the number of possible conformations in which lacosamide can interact with a sodium ion is unusually large. In the very diverse ensemble of lacosamide-binding modes, we found those that resemble more rigid ligands. In one of these modes, lacosamide chelated NaIII by two carbonyl oxygens, its aromatic ring bound between F 4i15 and Y 4i22 , and the NH groups approached oxygen atoms in positions p48 and p49 (Fig. 6 K).
In the obtained low-energy structures, sodium ion in position NaIII bound between two aromatic rings, enjoying cation-π interactions with both of them. The two hydroxyl groups anchored this complex to two backbone carbonyls p48 (Fig. 6, I and J). Despite in this binding mode, bisphenol A did not protrude an aromatic group in the inner pore, but it did contact F 4i15 . This result is consistent with the data that mutation F 4i15 A affects action of bisphenol A in the Nav1.5 channel (O’Reilly et al., 2012). Interestingly, bisphenol A, phenytoin, and lamotrigine have a common feature: a rigid or semi-rigid structure incorporating chemical groups that can donate protons to the backbone carbonyls at the opposing sides of the p48 ring.
Common and specific features of ligand-binding modes
Our calculations demonstrated that low-energy binding modes of cationic ligands in the NavMs-based model of Nav1.4 do not contradict the earlier predicted binding modes where the ligands’ cationic groups occur at the focus of P1 helices, close to putative site NaIV (Tikhonov and Zhorov, 2007 Bruhova et al., 2008). In addition, the NavMs-based model allowed us to find that different cationic ligands adopted a previously unknown binding mode in which the cationic group binds at site NaIII. When this site is occupied by a sodium ion, it attracts various electroneutral ligands, which also form multiple contacts with the channel protein. Within the groups of cationic and electroneutral ligands, the contacts with the channel are rather similar. Importantly, such contacts are also similar between the two groups. Fig. 7 shows superposition of the considered ligands. The cationic nitrogen or the sodium ion at one end of the ligand is highlighted blue or yellow, respectively, and the most remote atom in the aromatic ring at another end of the ligand is highlighted gray. Positions of these atoms, which largely determine the binding mode, are conserved with two exceptions. One is bisphenol A, which lacks an aromatic group protruding in the inner pore. The second exception is sipatrigine, an unusually long ligand. Besides these exceptions, the principal features of the ligand–channel complexes (Fig. 7 B) are consistent with the classical pharmacophore model for LAs, which is proposed by Khodorov (1981). The pharmacophore includes a cation (the ammonium group or ligand-bound sodium ion) and an aromatic moiety, which are usually linked by four bonds (Fig. 7 C). We expect that multiple compounds that are structurally related to the considered ones should also have analogous binding modes.
Scientists uncover hints of a novel mechanism behind general anesthetic action
Despite decades of common use for surgeries of all kinds, the precise mechanism through which general anesthesia works on the body remains a mystery. This may come as a surprise to the millions of Americans who receive inhaled general anesthesia each year. New research led by the Perelman School of Medicine at the University of Pennsylvania investigated the common anesthetic sevoflurane and found that it binds at multiple key cell membrane protein locations that may contribute to the induction of the anesthetic response. Their findings will appear online in PNAS (Proceedings of the National Academy of Science).
Previous studies have suggested that inhaled general anesthetics such as sevoflurane might work by inactivating sodium channels, specialized protein conduits that open in response to stimuli, like voltage changes, and allow sodium ions to cross the cell membranes of nerve cells. Despite the physiological importance of sodium channels and their possible role as general anesthetic targets, little is known about interaction sites or the mechanism of action.
Penn's Roderic Eckenhoff, MD, vice chair for Research and the Austin Lamont Professor of Anesthesiology and Critical Care leads a team of top medicine, chemistry, and biology researchers who were recently awarded an NIH grant to unravel the mysteries of anesthesia. This paper represents the team's most recent findings.
Researchers found that sevoflurane's interaction with sodium channels plays an essential role in the generation of the electrical impulses necessary for the communication between nerve cells in the brain. "We sought to understand the molecular basis of the interaction of sevoflurane with the sodium channel as a starting point to determine how similar anesthetics might elicit the anesthetic response," says the study's lead author, Annika Barber, PhD, a post-doctoral researcher in the department of Neuroscience at the Perelman School of Medicine at the University of Pennsylvania. At the time the research was conducted, she was a doctoral candidate at Thomas Jefferson University in Philadelphia.
In concert with the Institute for Computational Molecular Science of Temple University, Dr. Barber first used molecular dynamic simulation, a 3-D computer modeling method, to visualize possible interactions of sevoflurane with discrete parts of the bacterial sodium channel called NaChBac. This archetypal membrane protein is homologous to sodium channels found in human brain. "Given the physical and chemical properties of inhaled anesthetics, we expected binding to many possible sites simulation, however, helped us limit and identify the sites where the binding of sevoflurane might actually change the function of the sodium channel," explained Barber. The team found three key binding sites possibly linked to the anesthetic response. The first involves the channel's sodium pore itself, which is plugged by sevoflurane the second concerns the gate that governs opening and closing of the sodium channel in response to a voltage change across the membrane of a neuron and the third surrounds a second gate that controls sodium flow by changing the shape of the channel's narrow pore. These three sites, researchers hypothesize, work together to turn off firing of electrical impulses in key neurons and thus, induce the anesthetic state.
The Jefferson researchers validated the functional significance of these sites by directly measuring the activity of the sodium channel and conducting additional computer simulations. They found that low doses of sevoflurane made voltage-dependent activation of the sodium channel more favorable. This surprising action could explain the excitatory phase many patients experience during the onset of sevoflurane anesthesia. However, as the concentrations of the anesthetic increased, sevoflurane begins to block the sodium channel which might ultimately contribute to the state of anesthesia. These dose-dependent mutually antagonistic effects, in a single ion channel were surprising to the group, and emphasize the complexity of anesthetic action.
"Precisely how these interactions at one ion channel fit into the global effects of anesthesia remains to be seen," says Barber, and adds "this study paves the way to map relevant general anesthetic binding sites in sodium channels and helps understand how their modulation by sevoflurane might determine the physiological processes implicated in general anesthesia".
26.1 Body Fluids and Fluid Compartments
The chemical reactions of life take place in aqueous solutions. The dissolved substances in a solution are called solutes. In the human body, solutes vary in different parts of the body, but may include proteins—including those that transport lipids, carbohydrates, and, very importantly, electrolytes. Often in medicine, a mineral dissociated from a salt that carries an electrical charge (an ion) is called an electrolyte. For instance, sodium ions (Na + ) and chloride ions (Cl - ) are often referred to as electrolytes.
In the body, water moves through semi-permeable membranes of cells and from one compartment of the body to another by a process called osmosis. Osmosis is basically the diffusion of water from regions of higher concentration of water to regions of lower concentration of water, along an osmotic gradient across a semi-permeable membrane. As a result, water will move into and out of cells and tissues, depending on the relative concentrations of the water and solutes found there. An appropriate balance of solutes inside and outside of cells must be maintained to ensure normal function.
Body Water Content
Human beings are mostly water, ranging from about 75 percent of body mass in infants to about 50–60 percent in adult men and women, to as low as 45 percent in old age. The percent of body water changes with development, because the proportions of the body given over to each organ and to muscles, fat, bone, and other tissues change from infancy to adulthood (Figure 26.2). Your brain and kidneys have the highest proportions of water, which composes 80–85 percent of their masses. In contrast, teeth have the lowest proportion of water, at 8–10 percent.
Body fluids can be discussed in terms of their specific fluid compartment , a location that is largely separate from another compartment by some form of a physical barrier. The intracellular fluid (ICF) compartment is the system that includes all fluid enclosed in cells by their plasma membranes. Extracellular fluid (ECF) surrounds all cells in the body. Extracellular fluid has two primary constituents: the fluid component of the blood (called plasma) and the interstitial fluid (IF) that surrounds all cells not in the blood (Figure 26.3).
The ICF lies within cells and is the principal component of the cytosol/cytoplasm. The ICF makes up about 60 percent of the total water in the human body, and in an average-size adult male, the ICF accounts for about 25 liters (seven gallons) of fluid (Figure 26.4). This fluid volume tends to be very stable, because the amount of water in living cells is closely regulated. If the amount of water inside a cell falls to a value that is too low, the cytosol becomes too concentrated with solutes to carry on normal cellular activities if too much water enters a cell, the cell may burst and be destroyed.
The ECF accounts for the other one-third of the body’s water content. Approximately 20 percent of the ECF is found in plasma. Plasma travels through the body in blood vessels and transports a range of materials, including blood cells, proteins (including clotting factors and antibodies), electrolytes, nutrients, gases, and wastes. Gases, nutrients, and waste materials travel between capillaries and cells through the IF. Cells are separated from the IF by a selectively permeable cell membrane that helps regulate the passage of materials between the IF and the interior of the cell.
The body has other water-based ECF. These include the cerebrospinal fluid that bathes the brain and spinal cord, lymph, the synovial fluid in joints, the pleural fluid in the pleural cavities, the pericardial fluid in the cardiac sac, the peritoneal fluid in the peritoneal cavity, and the aqueous humor of the eye. Because these fluids are outside of cells, these fluids are also considered components of the ECF compartment.
Composition of Body Fluids
The compositions of the two components of the ECF—plasma and IF—are more similar to each other than either is to the ICF (Figure 26.5). Blood plasma has high concentrations of sodium, chloride, bicarbonate, and protein. The IF has high concentrations of sodium, chloride, and bicarbonate, but a relatively lower concentration of protein. In contrast, the ICF has elevated amounts of potassium, phosphate, magnesium, and protein. Overall, the ICF contains high concentrations of potassium and phosphate ( HPO 4 2 − HPO 4 2 − ), whereas both plasma and the ECF contain high concentrations of sodium and chloride.
Watch this video to learn more about body fluids, fluid compartments, and electrolytes. When blood volume decreases due to sweating, from what source is water taken in by the blood?
Most body fluids are neutral in charge. Thus, cations, or positively charged ions, and anions, or negatively charged ions, are balanced in fluids. As seen in the previous graph, sodium (Na + ) ions and chloride (Cl - ) ions are concentrated in the ECF of the body, whereas potassium (K + ) ions are concentrated inside cells. Although sodium and potassium can “leak” through “pores” into and out of cells, respectively, the high levels of potassium and low levels of sodium in the ICF are maintained by sodium-potassium pumps in the cell membranes. These pumps use the energy supplied by ATP to pump sodium out of the cell and potassium into the cell (Figure 26.6).
Fluid Movement between Compartments
Hydrostatic pressure , the force exerted by a fluid against a wall, causes movement of fluid between compartments. The hydrostatic pressure of blood is the pressure exerted by blood against the walls of the blood vessels by the pumping action of the heart. In capillaries, hydrostatic pressure (also known as capillary blood pressure) is higher than the opposing “colloid osmotic pressure” in blood—a “constant” pressure primarily produced by circulating albumin—at the arteriolar end of the capillary (Figure 26.7). This pressure forces plasma and nutrients out of the capillaries and into surrounding tissues. Fluid and the cellular wastes in the tissues enter the capillaries at the venule end, where the hydrostatic pressure is less than the osmotic pressure in the vessel. Filtration pressure squeezes fluid from the plasma in the blood to the IF surrounding the tissue cells. The surplus fluid in the interstitial space that is not returned directly back to the capillaries is drained from tissues by the lymphatic system, and then re-enters the vascular system at the subclavian veins.
Watch this video to see an explanation of the dynamics of fluid in the body’s compartments. What happens in the tissue when capillary blood pressure is less than osmotic pressure?
Hydrostatic pressure is especially important in governing the movement of water in the nephrons of the kidneys to ensure proper filtering of the blood to form urine. As hydrostatic pressure in the kidneys increases, the amount of water leaving the capillaries also increases, and more urine filtrate is formed. If hydrostatic pressure in the kidneys drops too low, as can happen in dehydration, the functions of the kidneys will be impaired, and less nitrogenous wastes will be removed from the bloodstream. Extreme dehydration can result in kidney failure.
Fluid also moves between compartments along an osmotic gradient. Recall that an osmotic gradient is produced by the difference in concentration of all solutes on either side of a semi-permeable membrane. The magnitude of the osmotic gradient is proportional to the difference in the concentration of solutes on one side of the cell membrane to that on the other side. Water will move by osmosis from the side where its concentration is high (and the concentration of solute is low) to the side of the membrane where its concentration is low (and the concentration of solute is high). In the body, water moves by osmosis from plasma to the IF (and the reverse) and from the IF to the ICF (and the reverse). In the body, water moves constantly into and out of fluid compartments as conditions change in different parts of the body.
For example, if you are sweating, you will lose water through your skin. Sweating depletes your tissues of water and increases the solute concentration in those tissues. As this happens, water diffuses from your blood into sweat glands and surrounding skin tissues that have become dehydrated because of the osmotic gradient. Additionally, as water leaves the blood, it is replaced by the water in other tissues throughout your body that are not dehydrated. If this continues, dehydration spreads throughout the body. When a dehydrated person drinks water and rehydrates, the water is redistributed by the same gradient, but in the opposite direction, replenishing water in all of the tissues.
Solute Movement between Compartments
The movement of some solutes between compartments is active, which consumes energy and is an active transport process, whereas the movement of other solutes is passive, which does not require energy. Active transport allows cells to move a specific substance against its concentration gradient through a membrane protein, requiring energy in the form of ATP. For example, the sodium-potassium pump employs active transport to pump sodium out of cells and potassium into cells, with both substances moving against their concentration gradients.
Passive transport of a molecule or ion depends on its ability to pass through the membrane, as well as the existence of a concentration gradient that allows the molecules to diffuse from an area of higher concentration to an area of lower concentration. Some molecules, like gases, lipids, and water itself (which also utilizes water channels in the membrane called aquaporins), slip fairly easily through the cell membrane others, including polar molecules like glucose, amino acids, and ions do not. Some of these molecules enter and leave cells using facilitated transport, whereby the molecules move down a concentration gradient through specific protein channels in the membrane. This process does not require energy. For example, glucose is transferred into cells by glucose transporters that use facilitated transport (Figure 26.8).
Disorders of the.
Fluid Balance: Edema
Edema is the accumulation of excess water in the tissues. It is most common in the soft tissues of the extremities. The physiological causes of edema include water leakage from blood capillaries. Edema is almost always caused by an underlying medical condition, by the use of certain therapeutic drugs, by pregnancy, by localized injury, or by an allergic reaction. In the limbs, the symptoms of edema include swelling of the subcutaneous tissues, an increase in the normal size of the limb, and stretched, tight skin. One quick way to check for subcutaneous edema localized in a limb is to press a finger into the suspected area. Edema is likely if the depression persists for several seconds after the finger is removed (which is called “pitting”).
Pulmonary edema is excess fluid in the air sacs of the lungs, a common symptom of heart and/or kidney failure. People with pulmonary edema likely will experience difficulty breathing, and they may experience chest pain. Pulmonary edema can be life threatening, because it compromises gas exchange in the lungs, and anyone having symptoms should immediately seek medical care.
In pulmonary edema resulting from heart failure, excessive leakage of water occurs because fluids get “backed up” in the pulmonary capillaries of the lungs, when the left ventricle of the heart is unable to pump sufficient blood into the systemic circulation. Because the left side of the heart is unable to pump out its normal volume of blood, the blood in the pulmonary circulation gets “backed up,” starting with the left atrium, then into the pulmonary veins, and then into pulmonary capillaries. The resulting increased hydrostatic pressure within pulmonary capillaries, as blood is still coming in from the pulmonary arteries, causes fluid to be pushed out of them and into lung tissues.
Other causes of edema include damage to blood vessels and/or lymphatic vessels, or a decrease in osmotic pressure in chronic and severe liver disease, where the liver is unable to manufacture plasma proteins (Figure 26.9). A decrease in the normal levels of plasma proteins results in a decrease of colloid osmotic pressure (which counterbalances the hydrostatic pressure) in the capillaries. This process causes loss of water from the blood to the surrounding tissues, resulting in edema.
Mild, transient edema of the feet and legs may be caused by sitting or standing in the same position for long periods of time, as in the work of a toll collector or a supermarket cashier. This is because deep veins in the lower limbs rely on skeletal muscle contractions to push on the veins and thus “pump” blood back to the heart. Otherwise, the venous blood pools in the lower limbs and can leak into surrounding tissues.
Medications that can result in edema include vasodilators, calcium channel blockers used to treat hypertension, non-steroidal anti-inflammatory drugs, estrogen therapies, and some diabetes medications. Underlying medical conditions that can contribute to edema include congestive heart failure, kidney damage and kidney disease, disorders that affect the veins of the legs, and cirrhosis and other liver disorders.
Therapy for edema usually focuses on elimination of the cause. Activities that can reduce the effects of the condition include appropriate exercises to keep the blood and lymph flowing through the affected areas. Other therapies include elevation of the affected part to assist drainage, massage and compression of the areas to move the fluid out of the tissues, and decreased salt intake to decrease sodium and water retention.
LOCAL ANESTHETIC PHARMACODYNAMICS
In clinical practice, LAs are typically described by their potency, duration of action, speed of onset, and tendency for differential sensory nerve block. These properties do not sort independently.
Potency and Duration
Nerve-blocking potency of LAs increases with increasing molecular weight and increasing lipid solubility. Larger, more lipophilic LAs permeate nerve membranes more readily and bind Na channels with greater affinity. For example, etidocaine and bupivacaine have greater lipid solubility and potency than lidocaine and mepivacaine, to which they are closely related chemically.
- Nerve-blocking potency of LAs increases with increasing molecular weight and increasing lipid solubility.
More lipid-soluble LAs are relatively water insoluble, highly protein bound in blood, less readily removed by the bloodstream from nerve membranes, and more slowly “washed out” from isolated nerves in vitro. Thus, increased lipid solubility is associated with increased protein binding in blood, increased potency, and longer duration of action. Extent and duration of anesthesia can be correlated with LA content of nerves in animal experiments. In animals, blocks of greater depth and longer duration arise from smaller volumes of more concentrated LA, compared with larger volumes of less-concentrated LA.
Speed of Onset
Many textbooks and review articles assert that the onset of anesthesia in isolated nerves slows with increasing LA lipid solubility and increasing pKa ( Table 2 ). At any pH, the percentage of LA molecules present in the uncharged form, largely responsible for membrane permeability, decreases with increasing pKa.However, of the two LAs with the fastest onset, etidocaine is highly lipid soluble and chloroprocaine has a pKa greater than that of other LAs. Finally, the LA rate of onset is associated with the aqueous diffusion rate, which declines with increasing molecular weight.
TABLE 2.Local anesthetic characteristics that tend to sort together.
Pharmacologic and Toxicologic
- Increasing potency
- Increasing onset time
- Increasing duration of action
- Increasing tendency for severe systemic toxicity
- In general, all tend to sort together
Differential Sensory Nerve Block
Regional anesthesia and pain management would be transformed by a LA that would selectively inhibit pain transmission while leaving other functions intact. However, sensory anesthesia sufficient for skin incision usually cannot be obtained without motor impairment. As was first demonstrated by Gasser and Erlanger in 1929, all LAs will block smaller (diameter) fibers at lower concentrations than are required to block larger fibers of the same type. As a group, unmyelinated fibers are resistant to LAs compared with larger myelinated A-δ fibers. Bupivacaine and ropivacaine are relatively selective for sensory fibers. Bupivacaine produces more rapid onset of sensory than motor block, whereas the closely related chemical mepivacaine demonstrates no differential onset during median nerve blocks ( Figure 10 ). True differential anesthesia may be possible when Nav isoform-selective antagonists become available. Certain Nav isoforms have been found to be prevalent in dorsal root ganglia, and (as previously noted) the relative populations of various Nav isoforms can change in response to various pain states.
Other Factors Influencing Local Anesthetic Activity
Many factors influence the ability of a given LA to produce adequate regional anesthesia, including the dose, site of administration, additives, temperature, and pregnancy. As the LA dose increases, the likelihood of success and the duration of anesthesia increase, while the delay of onset and tendency for differential block decrease. In general, the fastest onset and shortest duration of anesthesia occur with spinal or subcutaneous injections a slower onset and longer duration are obtained with plexus blocks.
- The effectiveness of a given LA is influenced by the dose, site of administration, additives, temperature, and changes in neural susceptibility, as seen during pregnancy.
Epinephrine is frequently added to LA solutions to cause vasoconstriction and to serve as a marker for intravascular injection. Epinephrine and other α1-agonists increase LA duration largely by prolonging and increasing intraneural concentrations of LAs. Blood flow is decreased only briefly, and the block will persist long after the α1-adrenergic effect on blood flow has dissipated. Other popular LA additions include clonidine, NaHCO3, opioids, dexamethasone, and hyaluronidase. Uncharged local anesthetics have greater apparent potency at basic pH, where an increased fraction of LA molecules is uncharged, than at more acidic pH ( Figure 11 ). Uncharged LA bases diffuse across nerve sheaths and membranes more readily than charged LAs, hastening onset of anesthesia. Some clinical studies showed that the addition of sodium bicarbonate had an inconsistent action during clinical nerve block however, not all studies demonstrated a faster onset of anesthesia. One might anticipate that bicarbonate would have its greatest effect when added to LA solutions to which epinephrine was added by the manufacturer. Such solutions are more acidic than “plain” (epinephrine-free) LA solutions to increase shelf life. Bicarbonate shortens the duration of lidocaine in animals. Curiously, once LAs gain access to the cytoplasmic side of the Na channel, H+ ions potentiate use-dependent block. Marked prolongation of local anesthesia can be achieved by incorporating LAs into liposomes, as has been done with bupivacaine in the some formulation.
Pregnant women and pregnant animals demonstrate increased neural susceptibility to LAs. In addition, spread of neuraxial anesthesia likely increases during pregnancy due to decreases in thoracolumbar cerebrospinal fluid volume.
BLOOD CONCENTRATIONS AND PHARMACOKINETICS
Peak LA concentrations vary by the site of injection ( Figure 12 ). With the same LA dose, intercostal blocks consistently produce greater peak LA concentrations than epidural or plexus blocks. As has been recently discussed by others, it makes little sense to speak of “maximal” doses of LAs except in reference to a specific nerve block procedure, since peak blood levels vary widely by block site. In blood, all LAs are partially protein bound, primarily to α1-acid glycoprotein and secondarily to albumin.
Affinity for α1-acid glycoprotein correlates with LA hydrophobicity and decreases with protonation (acidity). Extent of protein binding is influenced by the concentration of α1-acid glyco-protein. Both protein binding and protein concentration decline during pregnancy. During longer-term infusion of LA and LA-opioid combinations, concentrations of LA-binding proteins progressively increase There is considerable first-pass uptake of LAs by the lungs, and animal studies suggest that patients with right-to-left cardiac shunting may be expected to demonstrate LA toxicity after smaller intravenous bolus doses.
- Recommendations on maximal doses of LAs commonly found in pharmacology texts are not terribly useful in the practice of clinical regional anesthesia.
- The serum concentrations of LAs depend on the injection technique, place of injection, and addition of additives to the LA.
- Any recommendation on the maximal safe LA dose can be valid only in reference to a specific nerve block procedure.
Esters undergo rapid hydrolysis in blood, catalyzed by non-specific esterases. Procaine and benzocaine are metabolized to para-aminobenzoic acid (PABA), the species underlying anaphylaxis to these agents. Higher doses of benzocaine, typically for topical anesthesia for endoscopy, can lead to life threatening levels of methemoglobinemia. The amides undergo metabolism in the liver. Lidocaine undergoes oxidative N-deal-kylation (by the cytochromes CYP 1A2 and CYP 3A4 to monoethyl glycine xylidide and glycine xylidide). Bupivacaine, ropivacaine, mepivacaine, and etidocaine also undergo N-dealkylation and hydroxylation. Prilocaine is hydrolyzed to o-toluidine, the agent that causes methemoglobinemia. Prilocaine doses of as little as 400 mg in fit adults may be expected to produce methemoglobinemia concentrations great enough to cause clinical cyanosis. Amide LA clearance is highly dependent on hepatic blood flow, hepatic extraction, and enzyme function therefore, amide LA clearance is reduced by factors that decrease hepatic blood flow, such as β-adrenergic receptor or H2-receptor blockers, and by heart or liver failure. Disposition of amide LAs is altered in pregnancy due to increased cardiac output, hepatic blood flow, and clearance, as well as the previously mentioned decline in protein binding. Renal failure tends to increase volume of distribution of amide LAs and to increase the accumulation of metabolic by-products of ester and amide LAs. Theoretically, cholinesterase deficiency and cholinesterase inhibitors should increase the risk of systemic toxicity from ester LAs however, there are no confirmatory clinical reports. Some drugs inhibit various cytochromes responsible for LA metabolism however, the importance of cytochrome inhibitors varies depending on the specific LA species. β-Blockers and H2-receptor blockers inhibit CYP 2D6, which may contribute to reduced amide LA metabolism. Itraconazole has no effect on hepatic blood flow, but inhibits CYP 3A4 and bupivacaine elimination by 20%–25. Ropivacaine is hydroxylated by CYP 1A2 and metabolized to 2′,6′-pipecoloxylidide by CYP 3A4. Fluvoxamine inhibition of CYP 1A2 reduces ropiva-caine clearance by 70%. On the other hand, coadministration with strong inhibitors of CYP 3A4 (ketoconazole, itraconazole) has only a small effect on ropivacaine clearance.
DIRECT TOXIC SIDE EFFECTS
It is a common, but misguided, assumption that all LA actions, including toxic side effects, arise from interaction with voltage-gated Na channels. There is abundant evidence that LAs will bind many other targets aside from Na channels, including voltage-gated K and Ca channels, KATP channels, enzymes, N-methyl-D-aspartate receptors, β-adrenergic receptors, G-protein-mediated modulation of K and Ca channels, and nicotinic acetylcholine receptors. LA binding to any one or all of these other sites could underlie LA production of spinal or epidural analgesia and could contribute to toxic side effects.
Central Nervous System Side Effects
Local anesthetic CNS toxicity arises from selectively blocking the inhibition of excitatory pathways in the CNS, producing a stereotypical sequence of signs and symptoms as the LA concentration in blood gradually increases ( Table 3 ). With increased LA doses, seizures may arise in the amygdala. With further LA dosing, CNS excitation progresses to CNS depression and eventual respiratory arrest. More potent (at nerve block) LAs produce seizures at lower blood concentrations and at lower doses than less-potent LAs. In animal studies, both metabolic and respiratory acidosis decreased the convulsive dose of lidocaine.
TABLE 3.Progression of signs and symptoms of toxicity as the local anesthetic dose (or concentration) gradually increases.
- Ominous feelings
- Circumoral numbness
- Myoclonic jerks
- Cardiovascular collapse
In laboratory experiments, most LAs will not produce cardiovascular (CV) toxicity until the blood concentration exceeds three times that necessary to produce seizures however, there are clinical reports of simultaneous CNS and CV toxicity with bupivacaine ( Table 4 ). In dogs, supraconvulsant doses of bupivacaine more commonly produce arrhythmias than supraconvulsant doses of ropivacaine and lidocaine. LAs produce CV signs of CNS excitation (increased heart rate, arterial blood pressure, and cardiac output) at lower concentrations than those associated with cardiac depression. Hypocapnia reduces ropivacaine-induced changes in ST segments and left ventricular contractility.
- In laboratory experiments, most LAs will not produce CV toxicity until the blood concentration exceeds three times that necessary to produce seizures.
TABLE 4.Convulsive versus lethal doses of local anesthetics in dogs.
|Dose producing convulsions in all animals (mg/kg)||22||5||4|
|Dose producing lethality in all animals (mg/kg)||76||20||27|
Local anesthetics bind and inhibit cardiac Na channels (Nav 1.5 isoform). Bupivacaine binds more avidly and longer than lidocaine to cardiac Na channels. As previously noted, certain R(+) optical isomers bind cardiac Na channels more avidly than S(–) optical isomers. These laboratory observations led to the clinical development of levobupivacaine and ropivacaine. Local anesthetics inhibit conduction in the heart with the same rank order of potency as for nerve block. Local anesthetics produce dose-dependent myocardial depression, possibly from interference with Ca signaling mech-anisms within cardiac muscle. These anesthetics bind and inhibit cardiac voltage-gated Ca and K channels at concentrations greater than those at which binding to Na channels is maximal. The LAs bind β-adrenergic receptors and inhibit epinephrine-stimulated cyclic adenosine monophosphate (AMP) formation. In rats, the rank order for cardiac toxicity appears to be bupivacaine > levobupivacaine > ropivacaine. In dogs, lidocaine was the least potent, and bupivacaine and levobupivacaine were more potent than ropivacaine at inhibiting left ventricular function as assessed by echocardiography ( Table 5 ). In dogs, both programmed electrical stimulation and epinephrine resuscitation elicited more arrhythmias after bupivacaine and levobupivacaine than after lidocaine or ropivacaine administration. The mechanism by which CV toxicity is produced may depend on which LA has been administered. When LAs were given to the point of extreme hypotension, dogs receiving lidocaine could be resuscitated but required continuing infusion of epinephrine to counteract LA-induced myocardial depression. Conversely, many dogs receiving bupivacaine or levobupivacaine to the point of extreme hypotension could not be resuscitated. After bupivacaine, levobupivacaine, or ropivacaine, dogs that could be defibrillated often required no additional therapy. Similarly, in pigs, comparing lidocaine with bupivacaine, the ratio of potency for myocardial depression was 1:4, whereas it was 1:16 for arrhythmogenesis. The LAs produce dilation of vascular smooth muscle at clinical concentrations. Cocaine is the only LA that consistently produces local vasoconstriction.
- True immunologic reactions to LAs are rare.
- True anaphylaxis appears more common with ester LAs that are metabolized directly to PABA than other LAs.
- Accidental intravenous injections of LAs are sometimes misdiagnosed as allergic reactions.
- Some patients may react to preservatives, such as meth-ylparaben, included with LAs.
True immunologic reactions to LAs are rare. Accidental intravenous injections of LAs are sometimes misdiagnosed as allergic reactions. True anaphylaxis appears more common with ester LAs that are metabolized directly to PABA than to other LAs. Some patients may react to preservatives, such as methyl-paraben, included with LAs. Several studies have shown that patients referred for evaluation of apparent LA allergy, even after exhibiting signs or symptoms of anaphylaxis, almost never demonstrate true allergy to the LA that was administered. On the other hand, LA skin testing has an excellent negative predictive value. In other words, 97% of patients who fail to respond to LA skin testing will also not have an allergic reaction to the LA in a clinical setting.
TABLE 5.Efects of local anesthetics on indices of myocardial function measured in dogs.
|Local Anesthetic||LVEDP (EC50 for 125% base) (mcg/mL)||dP/dtmax (EC50 for 65% base) (mcg/mL)||%FS (EC50 for 65% base) (mcg/mL)|
|Bupivacaine||2.2 (1.2–4.4)||2.3 (1.7–3.1)||2.1 (1.47–3.08)|
|Levobupivacaine||1.7 (0.9–3.1)||2.4 (1.9–3.1)||1.3 (0.9–1.8)|
|Ropivacaine||4.0 (2.1–7.5) a/sup>||4.0 (3.1–5.2) b||3.0 (2.1–4.2) a/sup>|
|Lidocaine||6.8 (3.0–15.4) c||8.0 (5.7–11.0) d||5.5 (3.5–8.7) d|
During the 1980s, 2-chloroprocaine (at that time formulated with sodium metabisulfite at a relatively acidic pH) occasion-ally produced cauda equina syndrome following accidental large-dose intrathecal injection during attempted epidural administration. Whether the “toxin” is 2-chloroprocaine or sodium metabisulfite remains unsettled: 2-chloroprocaine is now being tested as a substitute for lidocaine in human spinal anesthesia, and a series of publications suggest that it may be safe and effective. At the same time, other investigators have linked neurotoxic reactions in animals to large doses of 2-chlo-roprocaine rather than to metabisulfite. There is also contro-versy about transient neurologic symptoms and persistent sacral deficits after lidocaine spinal anesthesia. The reports and the controversy have persuaded many physicians to abandon lidocaine spinal anesthesia. Unlike other spinal LA solutions, lido-caine 5% permanently interrupts conduction when applied to isolated nerves or to isolated neurons. This may be the result of lidocaine-induced increases in intracellular calcium and does not appear to involve Na channel blockade. While it is impossible to “prove safety,” multiple studies suggest that chloroprocaine or mepivacaine can be substituted for lidocaine for brief spinal anesthesias.
Treatment of Local Anesthetic Toxicity
Treatment of adverse LA reactions depends on their severity. Minor reactions can be allowed to terminate spontaneously. Seizures induced by LAs should be managed by maintaining a patent airway and by providing oxygen. Seizures may be ter-minated with intravenous midazolam (0.05–0.10 mg/kg) or propofol (0.5–1.5 mg/kg) or a paralytic dose of succinylcho-line (0.5–1 mg/kg), followed by ventilation with bag and mask (or tracheal intubation). LA CV depression manifested by moderate hypotension, may be treated by infusion of intravenous fluids and vasopressors (phenylephrine 0.5–5 μg/kg/min, norepinephrine 0.02–0.2 μg/kg/min, or vasopressin 40 μg IV). If myocardial failure is present, epinephrine (1–5 μg/kg IV bolus) may be required. When toxicity progresses to cardiac arrest, the guidelines for treatment of LA toxicity as developed by the American Society of Regional Anesthesia and Pain Medicine (ASRA) are reasonable, and certainly preferable to the chaotic resuscitation schemes identified in a national survey prior to publication of the guideline. It makes sense that amiodarone be substituted for lidocaine and, based on multi-ple animal experiments, that smaller, incremental doses of epinephrine be used initially rather than 1-mg boluses. Animal experiments and clinical reports demonstrate the remarkable ability of lipid infusion to resuscitate from bupivacaine-induced cardiac arrest ( Figure 13 ).Given the nearly nontoxic status of lipid infusion, one cannot make a convincing argument to withhold this therapy from a patient requiring resuscitation from LA intoxication. With unresponsive bupivacaine cardiac toxicity cardiopulmonary bypass should be considered. It appears that the threat from severe local anesthetic systemic toxicity may be on the decline, whether from better treatment or from changes in techniques. A minority would argue that the risk was overstated from the start, at least in experienced hands. Many practitioners believe that ultrasound guidance during peripheral nerve blocks has led to safer practices and less risk. While this view remains controversial, there are studies that support this belief.
The neurotransmitter serotonin (also known as 5-hydroxytryptamine or 5-HT) is a close molecular relative of dopamine that belongs to the family of neurotransmitters known as monoamines (Kandel et al. 2000). Like dopamine, serotonin is made by small discrete clusters of neurons located at the base of the brain (see figure 5B). These serotonergic neurons connect to other neurons located throughout the CNS, including neurons in the cerebral cortex and other forebrain structures. Thus, serotonin has the capacity to influence a variety of brain functions including sensations related to environmental stimuli, pain perception, learning and memory, and sleep and mood (Kandel et al. 2000). The role of serotonin in mood control has received considerable attention in both the laboratory and the clinic, as selective serotonin reuptake inhibitors (SSRIs) such as Prozac® are the most widely prescribed drugs for depression and other mood disorders (Brunton et al. 2005 Kandel et al. 2000).
The majority of serotonin actions in the brain occur via activation of GPCRs. The human brain has 15 serotonin-activated GPCRs that activate a wide variety of G-protein subtypes (Kitson 2007). Thus, serotonin can produce neuromodulatory effects that tend to either increase or decrease neuronal output. Different subtypes of these GPCRs are found on presynaptic and postsynaptic neuronal structures in different brain regions. By activating these receptors, serotonin can enhance or inhibit neurotransmitter release at certain synapses and can produce slow hyperpolarizing or depolarizing synaptic responses at others. Intracellular signaling via serotonin-activated GPCRs also can influence the function of intracellular enzymes as well as alter gene expression.
Serotonin also can activate a single type of LGIC-type neurotransmitter receptor, the so-called 5-HT3 receptor (Thompson and Lummis 2007). This receptor contains a channel that is permeable to positively charged cations and thus produces fast activation of neurons when bound to serotonin. One interesting facet of the action of this receptor is that often it resides on axon terminals that contain and release GABA. Thus, activation of these presynaptic 5-HT3 receptors leads to a rapid release of GABA that will then inhibit downstream neurons.
In addition to the use of SSRIs for neuropsychiatric therapy, as mentioned above, a variety of other therapeutic uses exist for serotonergic drugs. The SSRIs produce their actions via inhibition of the serotonin transporter protein, as their name implies. Buspirone is an anxiety-reducing drug that acts on the 5-HT-1A receptor (Barrett and Vanover 1993), and use of this drug has been suggested for other disorders. The 5-HT3 antagonists routinely are used to reduce nausea and vomiting resulting from chemotherapy and can be used for similar purposes following surgical anesthesia, and these drugs also have been used for treatment of irritable bowel syndrome (Thompson and Lummis 2007).
The brain serotonergic system also is the target of psychoactive drugs, including many that are illegal. The largest class of hallucinogenic drugs, including LSD, mescaline, and psilocybin, are all partial agonists of the 5-HT2 receptor subtypes, and the 5-HT2A receptor is implicated in the effects of these drugs (Fantegrossi et al. 2008). Several amphetamine derivatives, such as 3,4-methylenedioxymethamphetamine (MDMA, also known as ecstasy), alter serotonin transporter molecules and increase synaptic serotonin levels (McKenna and Peroutka 1990). This may account for their sensory-enhancing effects.
Acute alcohol has mixed effects on serotonergic transmission (reviewed in Lovinger 1997). A slowing of serotonergic reuptake is observed (Daws et al. 2006), but this does not appear to be because of impairment of the serotonin transporter targeted by SSRIs. Alcohol also potentiates the function of the 5-HT3 receptor (Lovinger 1999).
Chronic alcohol exposure has been demonstrated to interact with various aspects of serotonergic transmission that could alter anxiety and affect (reviewed in Lovinger 1997). Based on the well-known role of serotonin in neural mechanisms underlying mood and stress responses, pharmacotherapies aimed at the serotonergic system have long been touted as potential treatments for alcoholism (reviewed in Ait-Daoud et al. 2006). Treatments with SSRIs are efficacious in reducing alcohol intake in laboratory animals (LeMarquand et al. 1994 Pettinati et al. 1996) but have had mixed success in humans (Ait-Daoud et al. 2006 Naranjo and Kadlec 1991). It is possible that these drugs might work best in patients with comorbid depression. The 5-HT3 antagonist ondansetron reduces relapse in alcoholics, particularly in those with early onset (reviewed in Ait-Daoud et al. 2006).
Hyperexcitability and sensitization of sodium channels of dorsal root ganglion neurons in a rat model of lumber disc herniation
Low back pain and sciatica are the most common symptoms of patients with lumbar disc herniation (LDH). The pathophysiology of lumbocrural pain and sciatica is not fully understood. The aim of the present study was to define the membrane properties and activities of voltage-gated sodium channels of dorsal root ganglion (DRG) neurons in a rat model of LDH.
LDH was established by transplantation of autologous nucleus pulposus (NP) to lumbar 5 and 6 spinal nerves (L5–L6 DRG) of adult male rats. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) were measured 1 day before and through 35 days after transplantation of NP. Changes in expression of VGSCs were determined by western blotting. L5–L6 DRGs neurons innervating the hindpaw were labeled with DiI and acutely dissociated for measuring excitability and sodium channel currents under whole-cell patch clamp configurations.
NP transplantation significantly reduced the PWT and PWL in association with a significant reduction in rheobase and an increase in numbers of action potentials evoked by 2X and 3X rheobase current stimulation. Voltage-gated sodium current density was significantly enhanced in L5–L6 DRG neurons from LDH rats. The inactivation curve showed a leftward shift in LDH rats while activation curve did not significantly alter. However, NP transplantation remarkably enhanced expression of NaV1.7 and NaV1.8 in L5–L6 DRGs but not in T10–12 DRGs.
These data suggest that NP application produces pain-related behavior and potentiates sodium current density of DRG neurons, which is most likely mediated by enhanced expression of NaV1.7 and NaV1.8.