# Are Voltage Sensors Really Embedded in Muscarinic Receptors?
## 2 Discussion
[3H]ACh binding measured in untreated synaptoneurosomes (black lines, circles) and in synaptoneurosomes treated (grey lines, triangles) with the S-enantiomer of DPI (5 \(\upmu\)M) in the presence of 1 \(\upmu\)M TTX (\(\mathbf{A}\)) or with the R-enantiomer of DPI (5 \(\upmu\)M) (\(\mathbf{B}\)), are presented. The non-specific binding of [3H]ACh was measured in the presence of 1 \(\upmu\)M atropine. (Ref. [12]). The pertussis toxin (PTX)-sensitive trimetric Gi- and Go-proteins are activated by muscarinic agonists [1; 2; 5; 12; 14]. The activation of these G-proteins was measured in-situ in synaptoneurosomes by the covalent binding of a labeled GTP [14]. The covalent binding of labeled GTP to trimetric Go-proteins was induced by the binding of cholinergic receptor agonists to muscarinic receptors as well as in response to membrane depolarization [14]. Go-proteins are a highly conserved subtype of trimetric G-proteins, expressed in excitable tissues of numerous species [23]. Go-proteins are highly expressed in several regions of the mammalian brain and in peripheral nervous tissues. In addition, Go-proteins are highly expressed in the cardiac atria [23]. Unlike Gi-proteins, Go-proteins are activated in response to membrane depolarization, even in the absence of receptor stimulation [14]. Activation of Go-proteins was measured in-situ by tracing the covalent binding of [\(\alpha^{32}\)P]GTP-azidoanilide under UV-irradiation in depolarized synaptoneurosomes [14]. No covalent binding of [\(\alpha^{32}\)P]GTP-azidoanilide to Go proteins was measured at resting membrane potential [14]. Furthermore, [\(\alpha^{32}\)P]GTP-azidoanilide binding to Go-proteins in the depolarized synaptoneurosomes was not dependent on depolarization-induced stimulation of muscarinic receptors. Namely, [\(\alpha^{32}\)P]GTP-azidoanilide binding to Go-proteins in response to membrane depolarization was not abolished in the presence of antagonists of muscarinic cholinergic receptors. Neither was the depolarization-induced [\(\alpha^{32}\)P]GTP-azidoanilide binding to Go-proteins prevented by antagonists to additional receptors, including adrenergic, dopaminergic, and serotonergic receptors [14]. In contrast, the open configuration of VDSC in the depolarized synaptoneurosomes was indispensable for the depolarization-induced exchange of GDP by [\(\alpha^{32}\)P]GTP-azidoanilide in Go-proteins, even when Na\({}^{+}\) entry was blocked by TTX [14]. In addition, the \(\alpha\)-subunit of Go-proteins co-immunoprecipitationprecipitated with the \(\alpha\)-subunit of the VDSC in depolarized synaptoneurosomes [14], indicating a possible interaction between these proteins in the depolarized membranes [14]. Furthermore, the displacement of GDP by GTP in Go-proteins in response to membrane depolarization was accompanied by a depolarization-induced modulation of the high affinity binding of [\({}^{3}\)H]acetylcholine to muscarinic receptors from a high to a low affinity [14]. In addition, modifications preventing the activation of PTX-sensitive trimetric G-proteins interfered with the depolarization-induced activation of Go-proteins and with the depolarization-induced modulation of muscarinic receptors affinity for their cholinergic agonists [5; 12; 13; 14]. Thus, PTX-induced ADP-ribosylation of the \(\alpha\)-subunit of Go- and Gi-proteins, as well as the irreversible binding of GDP\(\beta\)S to trimetric G-proteins, prevented the activation of Go-proteins by muscarinic agonists as well as their activation by membrane depolarization [12; 14]. Furthermore, these modifications in G-proteins also prevented the depolarization-induced modulation of the high affinity of muscarinic receptors for [\({}^{3}\)H]acetylcholine into a low affinity [12; 14]. These findings associate the depolarization-induced exchange of GDP by [\(\alpha^{32}\)P]GTP-azidoanilide in activated Go-proteins with the depolarization-induced affinity modulation of muscarinic receptors for [\({}^{3}\)H]acetylcholine [12; 14].