# Are Voltage Sensors Really Embedded in Muscarinic Receptors?
## 2 Discussion
Furthermore, the open configuration of VDSC was a pre-requisite for the effect of G-protein activation on muscarinic affinity modulation (even when Na\({}^{+}\) entry was blocked) [12; 13; 14]. Thus, the effect of a permanent binding of GTP to G-proteins (Gpp(NH)p) keeping G-proteins activated on the affinity modulation of muscarinic receptors for [\({}^{3}\)H]acetylcholine was eliminated when the opening of VDSC was prevented [14]. A permanent binding of Gpp(NH)p reduced the high affinity of the muscarinic receptor for [\({}^{3}\)H]acetylcholine, but only when the VDSC were kept in their open configuration in synaptoneurosomes treated with BTX or the S-enantiomer of DPI (Refs. [12; 13; 14]; Figure 3). Preventing the open configuration of the VDSC by the binding of the R-enantiomer of DPI to the VDSC [20] prevented the effect of persistent activation of G-proteins on the high- to low-affinity modulation of muscarinic receptors for [\({}^{3}\)H]acetylcholine (Ref. [14]; Figure 3). According to these results, the open configuration of the VDSC was required for affinity modulation of muscarinic receptors by activation of G-proteins (Figure 3). Thus, since the affinity of muscarinic receptors for agonists is modulated by activation of PTX-sensitive Gi- and Go-proteins [14], and Go-proteins are activated by membrane depolarization, membrane-depolarization-induced activation of Go-proteins could modulate the affinity of muscarinic receptors for agonists (Figures 2 and 3). Furthermore, the depolarization-induced opening of VDSC was indispensable for the depolarization-induced activation of Go-protein as well as for the depolarization-induced affinity modulation of muscarinic receptors (Refs. [12; 13; 14]; Figures 2 and 3). Thus, according to these results, VDSC gating [17; 18; 19; 20; 21], acting as the voltage sensor generating activation of Go-proteins in response to membrane depolarization, causes the affinity modulation of the muscarinic receptor for cholinergic agonists in response to membrane depolarization. This mechanism is in accordance with the suggested role of loops L2 and L3 in the muscarinic receptor, which are implicated in the coupling of the muscarinic receptor with G-proteins and in the depolarization-induced affinity modulation of the muscarinic receptors [8; 9; 22]. Moreover, the indispensable role of the open configuration of VDSC in the depolarization-induced affinity modulation of muscarinic receptors for their cholinergic agonists [12; 13; 14] may explain the discrepancy between the measured gating current even when charges in loop L3 were missing and the attributed gating current to charge movement in the L3 loop of muscarinic receptors [9]. So, in addition to other identified inconsistencies [9; 11], an embedded charge movement in the muscarinic receptor [6; 8] does not seem to be in line with findings indicating that the depolarization-induced modifications in VDSC are indispensable for depolarization-induced modulation of the muscarinic receptor affinity for their cholinergic agonists (Refs. [12; 13; 14]; Figures 2 and 3). Notably, modifications in G-proteins preventing their activation did not interfere with muscarinic cholinergic agonists inducing the opening of VDSC at resting membrane potential [15]. This result may exclude the involvement of G-proteins' activation in the opening of VDSC by cholinergic agonists that bind with a high affinity to muscarinic receptors at resting membrane potential [12; 13; 14]. Thus, under physiological conditions, muscarinic  
Figure 3: The effect of a permanent binding of GTP (Gpp(NH)p) (200 \(\upmu\)M) on the high-affinity binding of [3H] acetylcholine ([3H] ACh) to muscarinic receptors in rat brainstem synaptoneurosomes is dependent on the configuration of VDSC in the presence of the R-enantiomer or the racemic mixture of the cardiotonic drug DPI. (**A**). [3H] ACh binding to muscarinic receptors when G-proteins bind Gpp(NH)p (\(\blacksquare\)) and in the absence of Gpp(NH)p (\(\blacksquare\)). (**B**).