Determining the target of membrane sterols on voltage-gated potassium channels.

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel Kv1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing Kv1.3 and Kv10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-ß-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.

Cytoplasmic Ca2+ concentration changes evoked by muscarinic cholinergic stimulation in primary and metastatic melanoma cell lines.

Experiments were performed to explore differences between cultured primary and metastatic melanoma cell lines in their muscarinic acetylcholine receptor-mediated intracellular Ca signalization. The expression of type 1 and type 3 muscarinic receptors was detected and compared at the protein level using both immunocytochemistry and semiquantitative western blotting. The functionality of muscarinic receptors was tested by applying carbamylcholine (CCh; 1 mmol/l) and by recording the associated increases in cytoplasmic Ca using Ca imaging with the application of the Ca indicator dye, fluo-4. These data indicate that the expression levels of the receptor proteins were not significantly different in the metastatic (HT199, HT168-M1) and the primary (WM35) cell lines. Although Ca transients were evoked in all the three cell lines by CCh, the proportion of the CCh-positive cells was smaller amongst the WM35 cells. The Ca transients could be effectively blocked by atropine (0.1 mmol/l). The time courses of the Ca transients were highly variable, and in some instances they showed a late (plateau-like) component whose presence crucially depended on the influx of extracellular Ca. When the extracellular Ca concentration was reduced, the duration of the CCh-evoked transients was considerably decreased; a phenomenon that was more pronounced in the metastatic cell lines. Although there are no fundamental differences in the muscarinic receptor-mediated Ca signalization of the primary and metastatic cell lines, the quantitative differences showed in this study may partially explain the increased malignancy and migratory potential of the metastatic cells.

Purkinje-like cells of the rat cochlear nucleus: a combined functional and morphological study.

Purkinje-like cells (PLCs) of the cochlear nucleus (CN) are strongly calbindin positive neurones with unknown function. In the present work functional and morphological methods have been employed to provide data about PLCs in general, and about their possible involvement in the synaptic organisation of the CN in particular. PLCs had slightly elongated soma, from which a complex dendritic arborisation extended with highly variable dimensions. On the basis of their morphology, three classes of PLCs were identified. Positively identified PLCs fired a train of action potentials on sustained depolarization. When hyperpolarizing stimuli were applied, the presence of a slowly activating, ZD7288-sensitive inward current was noted that corresponded to the h-current. PLCs received both excitatory and inhibitory synaptic inputs. Functional experiments revealed that 76% and 14% of the spontaneous inhibitory postsynaptic currents recorded from the cell bodies of the PLCs were mediated via glycinergic and GABAergic synapses, respectively. PLCs presented strong cerebellin1-like immunoreactivity, but its distribution differed from that seen in cerebellar Purkinje cells. Our results indicate that PLCs are parts of the synaptic circuitry of the CN, thus they may be actively involved in the processing and analysis of auditory information.

Cytoplasmic Ca(2+) concentration changes evoked by cholinergic stimulation in primary astrocyte cultures prepared from the rat cochlear nucleus.

The involvement of astrocytes in the cholinergic modulation of the cochlear nucleus has been studied using primary astrocyte cultures prepared from this nucleus. The cells were loaded with the membrane permeable form of the fluorescent Ca(2+) indicator Fluo-4, and carbachol-induced Ca(2+) concentration increases were monitored using an imaging system. In the presence of cholinergic stimulation 36.3% of the cells produced Ca(2+) transients. The time course of the transients was variable; 45.0% of the responding cells showed only a rapid Ca(2+) concentration increase, while in 50.5% of the astrocytes the fast component was followed by a slow plateau phase. Using muscarine as well as general and more specific cholinergic antagonists (atropine, pirenzepine, 4-DAMP and hexamethonium), the role of the M3 and (to a smaller extent) M1 muscarinic acetylcholine receptors could be demonstrated in the genesis of the carbachol-induced Ca(2+) transients. The presence of these two subtypes of muscarinic receptors has been confirmed at both mRNA (Q-PCR) and protein (immunocytochemistry) levels. Our data demonstrate the responsiveness of the cochlear astrocytes towards cholinergic stimulation, suggesting that they may have roles in mediating the effects of cholinergic modulation in the rat cochlear nucleus.

Rhodamine backfilling and confocal microscopy as a tool for the unambiguous identification of neuronal cell types: a study of the neurones of the rat cochlear nucleus.

Adequate interpretation of the functional data characterising the projection neurones of the cochlear nucleus (CN) is impossible without the unequivocal classification of these cell types at the end of the experiments. In this study, morphological criteria applicable for unambiguous identification of CN neurones have been sought. The neurones were labelled with rhodamine from incisions severing the projection pathways of the individual cell types, allowing their selective labelling and morphological characterisation. Confocal microscopy was employed for the investigation of the rhodamine-filled cells whose morphology was assessed after reconstructing the three-dimensional images of the cell bodies and proximal processes. The diameters of the somata and the number of processes originating from the cell bodies were also determined. In most of the cases, unambiguous identification of the bushy, octopus and Purkinje-like cells was relatively straightforward. On the other hand, precise classification of the pyramidal cells was often difficult, especially because giant cells could easily possess morphological features resembling pyramidal neurones. Occasionally, giant cells also mimicked the appearance of octopus neurones, which may be another important source of identification error, especially as these two cell types are often situated close to each other in the CN. It is concluded that morphological criteria defined in the present work may be effectively applied for the unambiguous identification of the projection neurones of the CN, even following functional measurements, when the correct cell classification is essential for the interpretation of the experimental data. Moreover, the present study also confirmed that Purkinje-like cells project to the cerebellum.