Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.The functional properties of nicotinic acetylcholine receptors (nAcChoRs) have been elucidated by many approaches: for example, electrophysiological recordings from muscle (1), measurements of ion fluxes in membrane vesicles containing nAcChoRs (2), expression of mRNAs encoding nAcChoRs in Xenopus oocytes (3)(4)(5), and, more recently, by injecting oocytes with membranes from Torpedo electroplaques (6). The latter approach results in the incorporation of native Torpedo nAcChoRs and other proteins into the oocyte's plasma membrane (6). This procedure has some important advantages over the usual oocyte expression system, since it allows the study of receptors that have been fully processed and assembled, with their natural subunit stoichiometry, in the original cell membrane. Nevertheless, there are many reasons that make the incorporation of well-defined purified proteins into the host cell membranes highly desirable. Therefore, we set to find out whether purified nAcChoRs reconstituted in a lipid matrix could be incorporated in a host cellular system such as the Xenopus oocyte, in which the function and cellular regulation of the protein can be examined in detail. Preliminary results have been presented elsewhere (7,8). METHODSSolubilization and Reconstitution of nAcChoRs. nAcChoRrich membranes from the electric organ of Torpedo marmorata were used to purify nAcChoRs by affinity chromatography in the presence of asolectin lipids and with cholate as detergent (9-11) (Fig. 1). The specific activity of the purified nAcChoRs was 'z-8 nmol of a-bungarotoxin bound per mg of protein (10).Reconstitution of nAcChoRs in asolectin lipid vesicles was accomplished by a detergent dialysis method (10). Final concentrations in the reconstitution mixtures were as follows: nAcChoRs, 0.3-1.2 mg of protein per ml; asolectin lipids, -5 mg/ml; and sodium cholate, 1% (wt/vol). After dialysis, reconstituted nAcChoR samples were aliquoted and injected immediately into oocytes or stored in liquid nitrogen, either alone or in the presence of trehalose (5 mg/mg of protein) to prevent protein denaturation (10).Oocyte Preparation and Microinjection. Xenopus ...

Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.

SUMMARY1. The properties of sensory neurones in the petrosal ganglion of the cat were examined in vitro with intracellular electrodes 8 days after section of the central (bulbar roots) or peripheral process. Two types of cells, both with conduction velocities faster than 2 m/s and with humps on the falling phases of their action potentials (H-neurones), were studied: glossopharyngeal neurones arising from the tongue and pharynx, and carotid neurones originating in the carotid body and carotid sinus.2. Peripheral axotomy produced an increase in action potential duration and a marked decrease in the amplitude and duration of the spike after-hyperpolarization in both glossopharyngeal and carotid neurones.3. The maximum rate of depolarization of the action potential increased after peripheral axotomy in glossopharyngeal cells but did not change in carotid neurones.4. The time-dependent inward rectification in response to hyperpolarizing pulses was markedly reduced in both types of cells after peripheral axotomy.5. Section of the peripheral process produced a decrease of the rheobase of glossopharyngeal cells, but not of carotid neurones. After axotomy the proportion of cells giving tonic discharges in response to long depolarizing pulses increased from 13 to 54% among carotid neurones but did not change in glossopharyngeal cells.6. No significant changes in membrane potential or input resistance of either group of cells were found after peripheral axotomy.7. Central axotomy did not produce any changes in the electrophysiological properties of glossopharyngeal or carotid neurones.8. Peripheral conduction velocity was decreased in both types of cells after peripheral axotomy, but did not change after section of the bulbar roots.9. It is concluded that the electrical properties of sensory neurones are modified after peripheral axotomy but not after central axotomy. Furthermore, the changes produced by peripheral axotomy are different in neurones innervating different peripheral targets.10. The possibility that some electrical properties of sensory neurones are maintained by their peripheral targets is discussed.

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

This work explores whether the ion selectivity and permeation properties of a model potassium channel, KcsA, could be explained based on ion binding features. Non-permeant Na or Li bind with low affinity (millimolar K's) to a single set of sites contributed by the S1 and S4 sites seen at the selectivity filter in the KcsA crystal structure. Conversely, permeant K, Rb, Tl and even Cs bind to two different sets of sites as their concentration increases, consistent with crystallographic evidence on the ability of permeant species to induce concentration-dependent transitions between conformational states (non-conductive and conductive) of the channel's selectivity filter. The first set of such sites, assigned also to the crystallographic S1 and S4 sites, shows similarly high affinities for all permeant species (micromolar K's), thus, securing displacement of potentially competing non-permeant cations. The second set of sites, available only to permeant cations upon the transition to the conductive filter conformation, shows low affinity (millimolar K's), thus, favoring cation dissociation and permeation and results from the contribution of all S1 through S4 crystallographic sites. The differences in affinities between permeant and non-permeant cations and the similarities in binding behavior within each of these two groups, correlate fully with their permeabilities relative to K, suggesting that binding is an important determinant of the channel's ion selectivity. Conversely, the complexity observed in permeation features cannot be explained just in terms of binding and likely relates to reported differences in the occupancy of the S2 and S3 sites by the permeant cations.