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.