# Are Voltage Sensors Really Embedded in Muscarinic Receptors?
## 2 Discussion  
Different affinities of muscarinic receptors for their agonists were identified four decades ago [4]. A depolarization-induced modulation of the affinity of muscarinic receptors for their cholinergic agonists was first identified in the rat brain cortex, brain stem, and atria [5]. It was further confirmed for the affinity of the M2 muscarinic receptor subtype [6]. The voltage-induced modulation of the muscarinic receptors affinity for agonists is apparently one of the common features of muscarinic receptor subtypes [4, 5, 7]. Recent studies attribute the voltage-dependent modulation of muscarinic receptor affinity to a voltage-induced charge movement in the muscarinic receptor [6, 8]. A charge movement producing gating current was attributed to modifications in the intramolecular loops L2 and L3 of muscarinic receptors, which are implicated in the coupling of the muscarinic receptor to trimetric G-proteins [6, 8]. Unlike previous reports, these studies attribute opposite effects of membrane depolarization on the affinity of muscarinic receptor subtypes M1 and M2 [5, 8]. M1 is the main muscarinic receptor subtype in the brain, and the M2 muscarinicreceptor subtype is most abundant in the atria [1]. A voltage-induced charge movement in the muscarinic receptors was attributed to charged residues in the L3 intramolecular loop of the muscarinic receptor [6; 8]. However, this idea was inconsistent with the affinity changes resulting from their removal [9]. Removal of charged residues from the amino-terminal end of loop L3 in M2 muscarinic receptors abolished their depolarization-induced modification [9], but did not abolish the associated gating current measured in response to membrane depolarization, as expected for a gating sensor [9]. Moreover, gating currents were measured in the M1 subtype of muscarinic receptors in response to membrane depolarization, despite the fact that M1 receptors lack the charged sequence in loop L3 [9]. Thus, the conclusion of a tight correlation between the binding of muscarinic receptors to trimetric G-proteins and their affinity modulation was accurate [5; 9], but it did not indicate any tight correlation between a voltage-dependent charge movement in muscarinic receptors and the voltage-dependent modulation of their affinity for agonists [9; 10; 11]. A different mechanism may underlie the effect of membrane potential on the affinity of muscarinic receptors. This mechanism accurately fits findings indicating the indispensable role of the voltage-dependent sodium channel (VDSC) gating (even when Na\({}^{+}\) entry is blocked) in the depolarization-induced modulation of the muscarinic receptor affinity for cholinergic agonists [12; 13]. According to these findings, depolarization-induced opening of VDSC is essential for a depolarization-induced activation of trimetric G-proteins, which modulate the affinity of muscarinic receptors for their cholinergic agonists from high affinity to low affinity [12; 13; 14]. These findings are briefly described. An interaction between VDSC and muscarinic receptors was identified when muscarinic cholinergic agonists induced the opening of VDSC in synaptoneurosomes prepared from the brain cortex or brain stem [15]. Synaptoneurosomes consist of a presynaptic, rescaled neuron (synaptosomes) attached to a rescaled postsynaptic neuron (neurosomes), both containing the original content of their cytoplasm. Synaptoneurosomes were first prepared and examined by the group of [PERSON] [16]. The effect of membrane potential on different receptors in synapses was examined in synaptoneurosomes [5; 12; 13; 14; 15]. In these experiments, binding of cholinergic agonists to muscarinic receptors induced a Tetrodotoxin (TTX)-sensitive \({}^{22}\)Na\({}^{+}\) uptake, which was blocked by the muscarinic receptor antagonist atropine (Ref. [15]; Figure 1).