Nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we have determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes), and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild type (WT) mice expressed (%): α3β4 (55), α3β4α5 (24), and α3β4β2 (21) nAChRs. nAChRs in β4 knockout (KO) mice were reduced to less than 15 % of controls and no longer contained the α5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between α5β4 KO and WT, suggesting that the reduced number of receptors in the KO did not impair transganglionic transmission. Deletions of α5 or β2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct α3β4 and α3β2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the α-conotoxins AuIB and MII. Our data - based on biochemical and functional experiments and several mouse KO models - clarifies and significantly extends previous observations on the function of nAChRs in heterologous system and the SCG.

BackgroundThe mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.Methodology/Principal FindingsThe cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.Conclusions/SignificanceThe stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Highlights d Calcium channels are organized in transient nanodomains d Ca V 2.1 channel mobility influences synaptic vesicle release d C-terminal splicing of Ca V 2.1 alters association with synaptic vesicles d Transient channel-scaffold interactions are relevant for shortterm plasticity

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Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors ( nAChR s) in the mouse superior cervical ganglion ( SCG ) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α 3 β 4, α 3 β 4 α 5, and α 3 β 4 β 2 subunits, respectively. Moreover, mice lacking β 4 subunits do not express α 5‐containing receptors but still express a small number of α 3 β 2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α 5 β 4‐ KO and α 5 β 2‐ KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials ( CAP s) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α 5 β 4‐ KO and α 5 β 2‐ KO ganglia, whereas the stimulation threshold for eliciting CAP s was significantly higher in α 5 β 4‐ KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α 5 β 4‐ KO ganglia ( IC 50 : 22.1 μ mol/L), followed by α 5 β 2‐ KO ( IC 50 : 126.7 μ mol/L) and WT ganglia ( IC 50 : 389.2 μ mol/L). Based on these data, we estimated an IC 50 of 568.6 μ mol/L for a receptor population consisting solely of α 3 β 4 α 5 receptors; and we estimated that α 3 β 4 α 5 receptors comprise 72% of nAChR s expressed in the mouse SCG . Similarly, by measuring the effects of hexamethonium on AC h‐induced currents in cultured SCG neurons, we found that α 3 β ...

Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4-containing receptors constitute about 20 % of hetero-oligomeric nAChRs in postnatal day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2-double knockout (KO) mice removes all α4-containing receptors, suggesting that in the postnatal mouse SCG, α4 co-assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up-regulated in the SCG of P3 α5β4-double KO mice, where they make up about 50 % of receptors that bind [3H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4-double KO mice maintained for 1-2 days in culture comprise < 10 % of α4β2 and > 90 % of α3β2, as determined by patch clamp recordings with α4β2- and α3β2-specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (± α5) represent about 62 % of receptors, whereas 17 % are α3β2β4, and 21 % are α4β2 (± α5) receptors.

Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, β2, α5β2 or α5β2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3β4, α3β4α5 and α3β4β2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3β4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3β4α5 receptors had the same conductance as α3β4 receptors, but differed from α3β4 receptors by having an increased channel open time and increased burst duration. By contrast, α3β4β2 receptors differed from α3β4 and α3β4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties – and hence the receptors themselves – in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment.

Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks.