A potential analgesic medicinal substance has been discovered, the ouabain–Ca2+ chelate complex (EO). As we have found, the specific EO binding to the Na,K-ATPase (NKA) in nanomolar concentrations triggers several signaling cascades in the nociceptive neuron, two of which have been discussed elsewhere. The docking results indicate that the molecular basis for the specificity of EO–NKA binding is the formation of two intermolecular ionic bonds between the chelated Ca2+ cation and two NKA carboxylate anion, Glu116 and Glu117. The third downstream EO-triggered NKA/Src/PKA/p38 MAPK/NF-κB signaling pathway, likely, controls the GAP43 gene expression, which results in this case in the neurite-inhibiting effect at the tissue level. The strong EO analgesic effect at both the spinal and supraspinal levels has been demonstrated in the formalin test. EO is a promising candidate for the role of a novel and safe analgesic, which might be particularly effective for the treatment of the tumor-associated pain syndromes due to its possible cytostatic function.

We have experimented with freshly isolated single DRG neurons from neonatal (P0-5) rats to study currents mediated by voltage dependent Na+ (Nav) channels. All experiments were performed using the whole-cell mode of patch-clamp electrophysiology and following the standard steps of this technique. However, in a subgroup of neurons, spontaneous events resembling neurotransmitter release were observed under conditions optimized for whole-cell patch-clamp recordings of INa. All events have a fast rise phase (similar to responses of receptor channels), but decay in a heterogeneous manner. The waveform of the event closely matches that of the response of the purinergic receptor P2X type to ATP. This new activity in neurons was observed at -60 mV and was facilitated during relatively strong hyperpolarization. Although spontaneous fluctuations, termed membrane potential instabilities, are described in DRG neurons, the observed inward currents at more hyperpolarized states are distinct and novel. The spontaneous heterogeneous activities could be relevant to the elucidation of pain mechanisms by distinct pharmacological tools.

Two short arginine-containing tripeptides, H-Arg-Arg-Arg-OH (TP1) and Ac-Arg-Arg-Arg-NH2 (TP2), have been shown by the patch-clamp method to modulate the NaV1.8 channels of DRG primary sensory neurons, which are responsible for the generation of nociceptive signals. Conformational analysis of the tripeptides indicates that the key role in the ligand-receptor binding of TP1 and TP2 to the NaV1.8 channel is played by two positively charged guanidinium groups of the arginine side chains located at the characteristic distance of ~9 Å from each other. The tripeptide effect on the NaV1.8 channel activation gating device has been retained when the N- and C-terminal groups of TP1 were structurally modified to TP2 to protect the attacking peptide from proteolytic cleavage by exopeptidases during its delivery to the molecular target, the NaV1.8 channel. As demonstrated by the organotypic tissue culture method, the agents do not affect the DRG neurite growth, which makes it possible to expect the absence of adverse side effects at the tissue level upon administration of TP1 and TP2. The data obtained indicate that both tripeptides can have great therapeutic potential as novel analgesic medicinal substances.

The present work continues our recent series of articles that aim to elucidate the ligand–receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11–12 Å. According to the previously suggested mechanism of ligand–receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors to NaV1.8 channels. This study elucidates the role of the rhamnosyl residue of ouabain in the activation of the Na,K-ATPase signaling function. The effects resulting from activation of Na,K-ATPase signaling by the Ca2+ chelate complex of ouabain (EO) are not manifested upon removal of the rhamnosyl residue, as demonstrated in viable cells by the highly sensitive patch-clamp and organotypic cell culture methods. Docking calculations show that the rhamnosyl residue is involved in five intermolecular hydrogen bonds with the Na,K-ATPase α1-subunit, which are fundamentally important for activation of the Na,K-ATPase signaling function upon EO binding. The main contribution to the energy of EO binding is provided by its steroid core, which forms a number of hydrogen bonds and hydrophobic interactions with Na,K-ATPase that stabilize the ligand-receptor complex. Another critically important role in EO binding is expected to be played by the chelated Ca2+ cation, which should switch on strong intermolecular ionic interactions between the EO molecule and two α1-Na,K-ATPase amino acid residues, Glu116 and Glu117.

Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the NaV1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH2, Ac-RER-NH2, Ac-RAAR-NH2, Ac-REAR-NH2, Ac-RERR-NH2, Ac-REAAR-NH2, Ac-PRERRA-NH2, and Ac-PRARRA-NH2 has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand–receptor ion–ion bonds between the attacking peptide molecules and the NaV1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH2, and in the range of 9–12 Å in the active peptide molecules.

The article reports the discovery of a novel signaling cascade opioid-like receptor → Na,K-ATPase/Src → NaV1.8 channel in the nociceptive neuron membrane. Triggering this cascade results in the modulation of its effector unit—the NaV1.8 channel activation gating device, whereas the Na,K-ATPase/Src complex performs the signal transducer function. The cascade has three targets. Their modulation by the attacking molecules may evoke an antinociceptive response at the peripheral level. The first target is the opioid-like receptor activated by a number of gamma-pyrone derivatives. The second target is the Na,K-ATPase/Src complex, with its transducer function controlled by ouabain at nanomolar (endogenous) concentrations. The third target is the NaV1.8 channel activation gating device modulated by arginine-containing short peptides. The article discusses a possible mechanism of ligand-receptor binding of the arginine-containing tripeptide Ac-RRR-NH2 to the NaV1.8 channel in the primary sensory neuron membrane. Extracellular application of the tripeptide is shown by the patch-clamp method to decrease the voltage sensitivity of NaV1.8 channels. Positively charged guanidinium groups of arginine side chains are supposed to play the key role in the ligand-receptor complex formation. The results of conformational analysis demonstrate that the distances between the guanidinium groups in the tripeptide molecule exceed 10 Å. The obtained data lead us to conclude that the studied tripeptide can bind to the NaV1.8 channel using the mechanism described earlier for a range of other short arginine-containing peptides. In view of the foregoing, the tripeptide Ac-RRR-NH2 is a promising analgesic.

A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for the generation of the nociceptive signal; therefore, gamma-pyrones have great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained from living neurons using very sensitive physiological methods and the results of quantum chemical calculations. This approach allows the correlation of the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind to two calcium cations. One of them is chelated by the carbonyl and hydroxyl functional groups, while the other forms a salt bond with the carboxylate anion. Calcium-bound gamma-pyrones have fundamentally different electrostatic properties from free gamma-pyrone molecules. These two calcium ions are key elements involved in ligand-receptor binding. It is very likely that ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor activate the latter. The calculated intercationic distance of 9.5 Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.

Local voltage clamping and organotypic cultures of nervous tissue were used to study the actions of a series of agents whose activity is associated with the functioning of the GABAergic and NOergic systems on slow sodium channels. GABA was found not to affect Na V 1.8 channel activity, in contrast to substance RGPU-260, a composition of L-arginine and mefebut (β-phenyl-γ-aminobutyric acid methyl ester). Synthetic substance RGPU-260, like its component mefebut, was shown by our data to be able to reduce the functional activity of Na V 1.8 channels, giving its use potential as a peripherally acting analgesic drug. Sodium nitroprusside also decreased the functional activity of these channels, though this effect was seen only at relatively high concentrations, while its simultaneous use with RGPU-260 did not lead to any increase in the action on slow sodium channels. Analysis of the resulting data suggested that Na V 1.8 channels located in the asynaptic membranes of primary sensory neurons are not controlled by the GABAergic or NOergic systems of the brain.

The possible mechanisms of ligand–receptor binding of arginine-containing tetrapeptides with the NaV1.8 channels in the primary sensory neuron were investigated. Ac-RERR-NH2 tetrapeptide, acting outside the neuronal membrane, was found to decrease voltage sensitivity of the examined channels. In contrast, the Ac-REАR-NH2 tetrapeptide did not exhibit the same effect. Conformational analysis was used to investigate the mechanisms of ligand–receptor binding of a number of studied short peptides; it suggested that positively charged guanidine side chains of two arginine residues played a key role in peptide binding. Another amino-acid residue (glutamic acid) should be located between these two arginine residues. Our calculations demonstrated that the mechanism of ligand–receptor binding could not be implemented if the distance between the guanidine groups in short peptide molecules was less than a defined threshold value. The results allow one to conclude that the Ac-RERR-NH2 tetrapeptide and several other peptides capable of binding with the NaV1.8 channel by the same molecular mechanism have the potential to become novel peripheral analgesic drugs.

Исследованы возможные механизмы лиганд-рецепторного связывания аргининсодержащих тетрапептидов с каналами NaV1.8 первичного сенсорного нейрона. Установлено, что тетрапептид Ac-RERR-NH2, действующий с наружной стороны нейрональной мембраны, снижает потенциалочувствительность исследуемых каналов. В отличие от этого тетрапептид Ac-REАR-NH2 не обладает такой способностью. Применение конформационного анализа для объяснения механизма лиганд-рецепторного связывания ряда коротких пептидов, исследованных нами в настоящей работе, позволило предположить, что ключевую роль здесь играют положительно заряженные гуанидиновые группы боковых цепей двух аргинильных остатков. Между этими остатками должен находиться еще один аминокислотный остаток, в нашем случае - глутаминовая кислота. Расчеты показывают, что механизм лиганд-рецепторного связывания не может быть реализован, когда указанные гуанидиновые группы в молекулах коротких пептидов оказываются на расстоянии, меньшем определенного порогового значения. Полученные данные позволяют заключить, что тетрапептид Ac-RERR-NH2, равно как и ряд других пептидов, способных к связыванию с каналом NaV1.8 по тому же механизму, могут претендовать на роль лекарственных субстанций анальгетиков периферического механизма действия.

The responses of primary sensory neurons to subnanomolar and nanomolar ouabain concentrations, corresponding to the endogenous ouabain (EO) concentration, were studied. Atomic force microscopy (AFM) studies showed that exposure to EO led to an increase in neuron stiffness. Studies using local voltage clamping showed that ligand-receptor binding of EO with the Na,K-ATPase/Src complex decreased the effective charge of the activatory gate system of Na V 1.8 channels. Furthermore, the EO-activated intracellular cascade in which the Na,K-ATPase/Src complex operates as a signal transducer was found to lead to a decrease in the fluorescence intensity of antibodies to Na V 1.8 channels, as demonstrated by confocal laser scanning microscopy. These results suggest that EO, triggering the transduction function of the Na,K-ATPase/Src complex and the corresponding intracellular signal cascade, is able to modulate the expression of the SCN10A gene, which produces Na V 1.8 channels, which are responsible for encoding nociceptive signals.

Our results suggest that endogenous ouabain triggers two different signaling processes. The first, fast process, modulates the activation gating device of the NaV1.8 channels, thereby reducing their functional activity. The second, slow process, decreases the density of NaV1.8 channels in the membrane of the primary sensory neuron. We assume that in this case, endogenous ouabain triggers a downstream cascade leading to a decrease in the expression of the SCN10A gene that produces NaV1.8 channels. It can be concluded that endogenous ouabain, when it interacts with the primary sensory neuron, performs important function of modulating functional activity of NaV1.8 channels. The practical result of the study was the assumption that the delivery of ouabain as a drug substance to the membrane of a nociceptive neuron in nanomolar concentration should lead to a safe and effective antinociceptive action of this agent at the organismal level.

Effects of a synthetic cyclic peptide on the primary sensory neuron responses are investigated. A decrease in the Na V 1.8 channel voltage sensitivity upon application of the decapeptide Ac–Cys 1 –Leu 2 –Pro 3 –Arg 4 –Glu 5 –Arg 6 –Arg 7 –Ala 8 –Gly 9 –Cys 10 –NH 2 (PIP10) containing a Cys 1 –Cys 10 intramolecular disulfide bridge was demonstrated by the patch-clamp method. The side chains of Arg 4 and Arg 6 residues are sterically unhindered and not involved in strong intramolecular interactions, as shown by the full semiempirical AM1 geometry optimization of the PIP10 molecule. The distance between the central carbon atoms of the guanidinium moieties of these arginine residues is close to 14 Å. According to our hypothesis, it is these positively charged functional groups that are responsible for binding of PIP10 due to intermolecular ion-ionic bonding with negatively charged groups of the Na V 1.8 channel molecule. The agent had no effect on the neurite growth of sensory neurons. The results obtained make it possible to suppose that the PIP10 decapeptide can be used as a safe and effective analgesic medicinal substance, given its capability of specifically modulating the Na V 1.8 channels playing a key role in primary sensory coding in the afferent unit of the nociceptive system.

The responses of the primary sensory neuron to the effect of subnanomolar and nanomolar concentrations of ouabain, which correspond to its endogenous concentrations (EO), were investigated. By the method of atomic force microscopy (AFM) it was found that the effect of EO led to an increase in the stiffness of the neuron. It was found using the patch-clamp method that due to the ligand-receptor binding of EO to the Na, K-ATPase/Src complex, the effective charge of the activation gating system of NaV1.8 channels decreases. It was also found that EO-activated triggering of the intracellular cascade, in which the Na, K-ATPase/Src complex acts as a signal transducer, leads to a decrease in the fluorescence intensity of antibodies to NaV1.8 channels, which was revealed using confocal laser scanning microscopy. The results obtained allowed us to suggest that EO, triggering the transducer function of the Na, K-ATPase/Src complex and the corresponding intracellular signaling cascade, is able to modulate the expression of the SCN10A gene, which produces the NaV1.8 channels responsible for encoding nociceptive signals.

In the primary sensory neuron, ouabain activates the dual mechanism that modulates the functional activity of Na V 1.8 channels. Ouabain at endogenous concentrations (EO) triggers two different signaling cascades, in which the Na,K-ATPase/Src complex is the EO target and the signal transducer. The fast EO effect is based on modulation of the Na V 1.8 channel activation gating device. EO triggers the tangential signaling cascade along the neuron membrane from Na,K-ATPase to the Na V 1.8 channel. It evokes a decrease in effective charge transfer of the Na V 1.8 channel activation gating device. Intracellular application of PP2, an inhibitor of Src kinase, completely eliminated the effect of EO, thus indicating the absence of direct EO binding to the Na V 1.8 channel. The delayed EO effect probably controls the density of Na V 1.8 channels in the neuron membrane. EO triggers the downstream signaling cascade to the neuron genome, which should result in a delayed decrease in the Na V 1.8 channels’ density. PKC and p38 MAPK are involved in this pathway. Identification of the dual mechanism of the strong EO effect on Na V 1.8 channels makes it possible to suggest that application of EO to the primary sensory neuron membrane should result in a potent antinociceptive effect at the organismal level.

The aim of the study was to elucidate the molecular mechanisms of modulation of the NaV1.8 channels with a synthetic tetrapeptide (Ac-RERR-NH2). Our data suggest that this substance specifically modulates the activation gating device of these channels, which are responsible for coding of pain signals. This agent (0.1 nM) has a neurite-stimulating effect, which indicates its possible physiological regeneration effect on the nervous tissue. The results obtained allow us to conclude that the agent under study can claim to be the drug substance of a safe and effective analgesic.