Non-technical summary Muscarinic receptors were recently shown to be modulated by membrane potential. Here, we show that membrane potential alters the binding of agonists in an agonist-specific manner. Moreover, agonist binding results in agonist-specific conformational changes in the muscarinic receptor, as measured by changes in the receptor's response to voltage. Voltage-dependent modulation of muscarinic receptors has important consequences for cellular signalling in excitable tissues and implications for cardiovascular drug development.Abstract The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K + current, I KACh . Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I KACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist-receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.

The Cys-loop family of neurotransmitter-gated ion channels mediate excitatory and inhibitory transmission at fast chemical synapses. These proteins form pentameric assemblies with an extracellular ligand-binding domain, a transmembrane ion channel formed from 4 a-helices per subunit, and a large cytoplasmic globular domain between the M3 and M4 segments. The homopentameric proton-gated cation channel from the prokaryote Gloeobacter violaceus (Glvi) has been identified as a putative homologue but lacks some classical features of the eukaryotic family members, including the large cytoplasmic loop and the signature disulfide linkage. To gain insight into the structure of Glvi, we engineered 30 individual cysteine substitutions that align with positions -2' -27' of the M2 segment in eukaryotic family members. Each mutant was expressed in Xenopus oocytes and evaluated for the accessibility of the engineered cysteine to p-chloromercuribenzenesulfonate (pCMBS -) in the closed state (pH 7.5) or in the sub-maximally open state (pH 5). Of the 30 Cys mutants, in 5 the proton-induced currents were not significantly different than those in water-injected oocytes, and the 8' and 9' mutants showed aberrant gating properties. Of the mutants tested from -2' to 9', E220C (-2') and T224C (2') were reactive at both pH 7.5 and 5.0, and T229C (7') was reactive only at pH 5.0, presumably in the open state. However, from 10' to 27' each mutant tested was modified by pCMBSexcept V240C (19'). This suggests that the M2 helix is tightly associated with the adjacent transmembrane helices on the channel's intracellular side but is loosely packed from 10 0 to the extracellular end. Examination of the relative reaction rates may distinguish the channel-lining residues that are more extracellular than 10 0 .

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Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, Kir2.1, and the channel encoding the cardiac fast sodium current, Nav1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.