Mechanisms Underlying the Strong Inhibition of Muscle-Type Nicotinic Receptors by Tetracaine

Abstract

Nicotinic acetylcholine (ACh) receptors (nAChRs) are included among the targets of a variety of local anesthetics, although the molecular mechanisms of blockade are still poorly understood. Some local anesthetics, such as lidocaine, act on nAChRs by different means through their ability to present as both charged and uncharged molecules. Thus, we explored the mechanisms of nAChR blockade by tetracaine, which at physiological pH is almost exclusively present as a positively charged local anesthetic. The nAChRs from Torpedo electroplaques were transplanted to Xenopus oocytes and the currents elicited by ACh (IAChs), either alone or co-applied with tetracaine, were recorded. Tetracaine reversibly blocked IACh, with an IC50 (i.e., the concentration required to inhibit half the maximum IACh) in the submicromolar range. Notably, at very low concentrations (0.1 μM), tetracaine reduced IACh in a voltage-dependent manner, the more negative potentials produced greater inhibition, indicating open-channel blockade. When the tetracaine concentration was increased to 0.7 μM or above, voltage-independent inhibition was also observed, indicating closed-channel blockade. The IACh inhibition by pre-application of just 0.7 μM tetracaine before superfusion of ACh also corroborated the notion of tetracaine blockade of resting nAChRs. Furthermore, tetracaine markedly increased nAChR desensitization, mainly at concentrations equal or higher than 0.5 μM. Interestingly, tetracaine did not modify desensitization when its binding within the channel pore was prevented by holding the membrane at positive potentials. Tetracaine-nAChR interactions were assessed by virtual docking assays, using nAChR models in the closed and open states. These assays revealed that tetracaine binds at different sites of the nAChR located at the extracellular and transmembrane domains, in both open and closed conformations. Extracellular binding sites seem to be associated with closed-channel blockade; whereas two sites within the pore, with different affinities for tetracaine, contribute to open-channel blockade and the enhancement of desensitization, respectively. These results demonstrate a concentration-dependent heterogeneity of tetracaine actions on nAChRs, and contribute to a better understanding of the complex modulation of muscle-type nAChRs by local anesthetics. Furthermore, the combination of functional and virtual assays to decipher nAChR-tetracaine interactions has allowed us to tentatively assign the main nAChR residues involved in these modulating actions.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors

“…Furthermore, 5 µM Pm elicited a greater nAChR blockade and faster I ACh decay as compared to 1 µM Pm (1.18 ± 0.05 s against 0.83 ± 0.06 s for I ACh decay at 1 and 5 µM Pm, respectively; p = 0.002, paired t -test, same cells as above). As previously reported [ 26 ], deactivation of control I ACh s, elicited by ACh washout, followed an exponential function with a time course of roughly 1.5 s (this value likely limited by the solution exchange kinetics [ 26 ]).…”

“…These τ values were rather slow, as compared to those previously reported, under similar experimental conditions, for I ss inhibition elicited by different local anesthetics (LAs), as tetracaine or benzocaine, at their IC 50 . In fact, for these latter compounds, τ values were faster than the solution exchange kinetics (roughly 1.5 s [ 26 , 28 ]). Furthermore, the voltage-dependent blockade of nAChRs by Pm allowed us to determine more accurately the rate of open-channel blockade, by jumping the membrane potential during the I ACh elicited in the presence of Pm.…”

“…In agreement with this, the docking assays predicted some Pm clusters located within the channel pore, both in the open and closed conformations ( Figure 9 ). Thus, the open-channel blockade of nAChRs mediated by Pm resembles that mediated by some LAs, such as lidocaine [ 24 ] or tetracaine [ 26 ]. Nevertheless, the kinetics of open-channel blockade of nAChR elicited by Pm was rather slow.…”

“…Thus, at −60 mV, the τ of open channel blockade elicited by 1 µM Pm (close to its IC 50 ) was over 2 s and even by 5 µM Pm (eliciting roughly 70% of I ss blockade) the time constant was above 1 s ( Figure 7 ). In contrast, at the same membrane potential, 0.7 µM tetracaine (close to its IC 50 ) blocked open nAChRs with a time course of roughly 300 ms [ 26 ]. These differences in time constants between Pm and tetracaine are most likely related to their distinct molecular sizes (Pm molecular weight is over 60% greater than that of tetracaine).…”

Anesthetics: from modes of action to unconsciousness and neurotoxicity

“…Ϫ (Quasthoff et al 1995) Ϫ (Wen et al 2013) Ϫ (Quasthoff et al 1995;Sugiyama and Muteki 1994) Ϫ (Sugiyama and Muteki 1994) Ϫ (Gentry and Lukas 2001) Ϫ (Cobo et al 2018) Ϫ (Yost and Dodson 1993) Labeled targets are all inhibited (Ϫ) by the respective local anesthetics. K 2P , 2-pore domain K ϩ channel; nAChR, nicotinic acetylcholine receptor; VGCC, voltage-gated Ca 2ϩ channels; VGSC, voltage-gated Na ϩ channels.…”