Presynaptic cholinergic receptors and the role of calcium influx in the mechanism of ACh release autoinhibition
Since the inhibitory effect of nicotine on the QC was completely abolished by application of the antagonist of neuronal cholinergic receptors DHβE, it was concluded that these receptors are involved in the cholinergic mechanism of regulation of ACh release. DHβE binds to β2 subunits of neuronal receptors and is a selective antagonist for non-α7 nACh receptors (Stauderman et al., 2000). In the heteromeric receptors of ganglionic neurons the primary α subunit is α3, whereas in the rodent central nervous system the primary α subunit is α4 (Papke et al., 2010). The α4β2 nNAChR is the most abundant subtype expressed in the brain, and studies have demonstrated that this receptor subtype is located presynaptically (Karadsheh et al., 2004). Therefore, we can assume that in the neuromuscular synapse neuronal cholinergic receptors have the α4β2 subunit composition. This assumption is supported by the data that at 1 μM DHβE, that we used in this study, the mouse α4β2 receptors are almost completely blocked, while the α3β4 subunit receptors remain essentially active (Papke et al., 2010).
In the next step of the mechanism of autoinhibition triggered by nNAChRs, it was necessary to answer the following key question: how is the activation of the presynaptic nicotinic receptors coupled to changes in the intracellular calcium level? Previous data (Tian et al., 1994; Prior & Singh, 2000; Wang et al., 2018) were indicating such a coupling, but there was no direct evidence found for this prior to our study. Using of standard electrophysiological methods, combined with the fluorescent method for registration of calcium transients, which reflect changes in the calcium level within the presynaptic terminal upon action potential arrival, enabled us to obtain the data on changes in the ACh. Our results demonstrate that activation of nNAChRs (sensitive to DHβE), leading to a decrease in ACh release, is accompanied by an increase in the level of calcium in the nerve terminal. Another important observation made was a significant effect of DHβE on the amplitude of the calcium transient when applied alone. This may indicate that there exists a background tonic activation of nNAChRs which results in a tonic increase in calcium entry into the nerve terminal.
The complete absence of the effect of nicotine on the calcium signal after pre-treatment with cadmium (10 μM), which is a nonselective blocker of all types of calcium Cav channels (Hess et al., 1984) allows two conclusions to be put forward: (i) the increase in calcium level in the nerve terminal is mediated by transmembrane proteins permeable for Ca2+ from the environment; (ii) the observed entry of Ca2+ is mediated by channels other than those of nNAChRs. The last suggestion is very important, because it has been shown earlier that nNAChRs are more permeable to Ca2+ as compared to permeability of their muscle-type counterparts (Radford Deckera & Dani, 1990; Gotti & Clementi, 2004). At the same time, it was shown that cadmium up to a concentration of 200 μM does not block currents through nNAChRs (Wheeler et al., 2006), but significantly blocks currents through VGCCs (Lansman et al., 1986; Samigullin, Khaziev, Zhilyakov, Sudakov, et al., 2017).
After inactivation of VGCCs of Cav2.1 type, which are key to triggering the process of evoked ACh release (Nachshen & Blaustein, 1979; Protti & Uchitel, 1993; Katz et al., 1996) the effect of nicotine on calcium entry into the terminal was preserved, while after blockade of Cav1 type channels, it was completely abolished. It should be noted that the possibility of the involvement of these channels in the evoked release of ACh quanta remained under debate until recent times: (Prior & Singh, 2000; Urbano et al., 2002; Pagani et al., 2004; Perissinotti et al., 2008) versus (Penner & Dreyer, 1986; Atchison, 1989; Protti et al., 1991; Bowersox et al., 1995). We have obtained a clear evidence of the involvement of the Cav1 type of calcium channels in regulation of bulk calcium level in the terminal and of process of neurotransmission in the mammalian neuromuscular junction.