# Are Voltage Sensors Really Embedded in Muscarinic Receptors?
## 2 Discussion
In addition, specific binding of toxins to sites in the open configuration of VDSC, including the specific binding of labeled Batrachotoxin ([3H]BTX), was induced at resting membrane potential by the binding of cholinergic agonists to muscarinic receptors in the synaptoneurosomes. Their displacement by atropine prevented the induced [3H]BTX binding by muscarinic agonists (Refs. [13; 15]; Figure 1). Figure 1: TTX-blockable \({}^{22}\)Na\({}^{+}\) uptake (circles) and [3H]BTX binding (squares) to pertussis toxin-treated synaptoneurosomes, as a function of carbamylcholine concentration in the presence (\(\mathsf{O}\), \(\mathsf{\overline{O}}\)) and absence (\(\mathsf{\overline{O}}\), \(\mathsf{\overline{O}}\)) of atropine (0.1 \(\mathsf{uM}\)). The specific carbamylcholine-induced binding of [3H]BTX was measured in the presence of TTX (1 \(\mathsf{uM}\)). The non-specific carbamylcholine-induced \({}^{22}\)Na\({}^{+}\) uptake, both in the presence and absence of atropine, was measured in the presence of TTX (1 \(\mathsf{uM}\)) [15]. The binding of certain toxins to the open configuration of VDSC either keeps the VDSC in their open configuration (e.g., Batrachotoxin (BTX) and the S-enantiomer of the cardio-tonic drug DPI) or prevents the opening of VDSC (e.g., the R-enantiomer of the cardio-tonic drug DPI) [13; 17; 18; 19; 20; 21]. Muscarinic cholinergic receptor agonists dose-dependently enhanced the specific binding of [3H]BTX to open VDSC in the synaptoneurosomes at resting membrane potential, while Na\({}^{+}\) entry was blocked (Refs. [13; 15]: Figure 1). Thus, these findings suggest that binding of cholinergic receptor agonists to muscarinic receptors induces the opening of VDSC at resting membrane potential and that the \({}^{22}\)Na\({}^{+}\) entry is blocked by TTX, which blocks sodium currents via VDSC in the brain (Ref. [15]: Figure 1). A reciprocal effect of the VDSC gating on the affinity of muscarinic receptors for their agonists was identified as well [12; 13]. The high affinity binding of [3H]acetylcholine to muscarinic receptors in the brain synaptoneurosomes was substantially reduced under membrane depolarization [12; 13], and the open configuration of VDSC was indispensable for this voltage-induced affinity change in the muscarinic receptors, even when Na\({}^{+}\) entry was blocked [12; 13; 14]. Thus, a depolarization-induced reduction in the high-affinity binding of [3H]acetylcholine to muscarinic receptors was not measured when the opening of VDSC was prevented by the binding of the R-enantiomer of the cardiotonic drug DPI to the VDSC [12; 13; 14; 20] (Figure 2). According to these results, the open configuration of the VDSC (but not the Na\({}^{+}\) current) was indispensable for the depolarization-induced modulation of the muscarinic receptors' affinity for cholinergic agonists, from a high affinity to a low affinity (Refs. [12; 13; 14]: Figure 2). The binding of antagonists to muscarinic receptors was not similarly affected by membrane depolarization or by the open configuration of the VDSC [5; 12; 13]. Many findings identified the involvement of muscarinic receptor coupled trimeric G-proteins in the depolarization-induced affinity modulation of muscarinic receptors [5; 8; 9; 12; 14; 22]. One of these results showed that the L2 and L3 loops in the muscarinic receptor are implicated both in the receptor coupling to trimetric G-proteins and in the depolarization-induced modulation of its affinity for agonists [8; 9]. These findings are in accordance with the indispensable role of the depolarization-induced activation of trimetric G-proteins in the depolarization-induced modulation of the muscarinic receptors' affinity for their cholinergic receptor agonists [14]. Figure 2: Effects of DPI-enantiomers on the high affinity binding of [3H]acetylcholine ([3H]ACh) to muscarinic receptors in rat brain synaptoneurosomes at resting membrane potential (\(\mathbf{O}\), \(\triangle\)) and upon depolarization (\(\bullet\), \(\triangle\)).