The cellular membrane is very heterogenous and enriched with high-density regions forming microdomains, as revealed by single particle tracking experiments. However the organization of these regions remain unexplained. We determine here the biophysical properties of these regions, when described as a basin of attraction. We develop two methods to recover the dynamics and local potential wells (field of force and boundary). The first method is based on the local density of points distribution of trajectories, which differs inside and outside the wells. The second method focuses on recovering the drift field that is convergent inside wells and uses the transient field to determine the boundary. Finally, we apply these two methods to the distribution of trajectories recorded from voltage gated calcium channels and phospholipid anchored GFP in the cell membrane of hippocampal neurons and obtain the size and energy of high-density regions with a nanometer precision.

Organization of signalling molecules in biological membranes is crucial for cellular communication. Many receptors, ion channels and cell adhesion molecules are associated with proteins important for their trafficking, surface localization or function. These complexes are embedded in a lipid environment of varying composition. Binding affinities and stoichiometry of such complexes were so far experimentally accessible only in isolated systems or monolayers of cell culture. Visualization of molecular dynamics within signalling complexes and their correlation to specialized membrane compartments demand high temporal and spatial resolution and has been difficult to demonstrate in complex tissue like brain slices. Here we demonstrate the feasibility of single-particle tracking (SPT) in organotypic brain slices to measure molecular dynamics of lipids and transmembrane proteins in correlation to synaptic membrane compartments. This method will provide important information about the dynamics and organization of surface molecules in the complex environment of neuronal networks within brain slices.

We applied single-molecule fluorescence microscopy (using organic dyes or semiconductor quantum dots) to study the lateral diffusion of glutamate receptors (AMPA and NMDA) in live synapses. We directly imaged glutamate receptors movements inside and outside synapses of live cultured hippocampal neurons. We could record exchanges of receptors through lateral diffusion between these different membrane compartments. In addition, our data suggest that this lateral diffusion might be regulated by neuronal activity. To overcome the photobleaching problem inherent to fluorescence techniques we recently developed new optical methods for the detection of individual metallic nanoparticles. We can now detect signatures of diffusing AMPA receptors labeled with 10 nm gold nanoparticles on live neurons.

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.

CA1 pyramidal neurons of the hippocampus express various types of serotonin (5-HT) receptors, such as 5-HT1A, 5-HT4 and 5-HT7 receptors, which couple to Galpha(i) or Galpha(s) proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization-activated current I-h . Activation of 5-HT1A receptors induced an augmentation of current-induced hyperpolarization responses, while the responses declined after 5-HT4 receptors were activated. The resting potential of neurons hyperpolarized (-2.3 +/- 0.7 mV) after 5-HT1A receptor activation, activation of 5-HT4 receptors depolarized neurons (+3.3 +/- 1.4 mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the I-h current was identified by its characteristic voltage- and time-dependency and by blockade with CsCl or ZD7288. Activation of 5-HT1A receptors reduced I-h and shifted the activation curve to a more negative voltage by -5 mV at half-maximal activation. Activation of 5-HT4 and 5-HT7 receptors increased I-h and shifted the activation curve to the right by +5 mV. Specific activation of 5-HT4 receptors by BIMU8 increased membrane conductance and showed an increase in I-h in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5-HT4A receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5-HT7 receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.

The dynamics of intracellular free Ca2+([Ca2+]i) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca2+channels caused a steep increase in [Ca2+]i. Depolarizations lasting for < 100ms led to Ca2+release from intracellular stores as is indicated by the finding that the rise of [Ca2+]iwas greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca2+current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca2+influx enhanced in this way again caused a rise of [Ca2+]isensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca2+responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca2+responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca2+rises after having been treated with NHD. Thus, the variability of Ca2+signals might be caused by different filling states of Ca2+stores, and the resting Ca2+current seems to represent a source to fill empty Ca2+stores. In line with this notion, block of the endoplasmic Ca2+pump by thapsigargin (1μM) produced either no or largely varying Ca2+responses. The Ca2+signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca2+release channels. Block of mitochondrial Ca2+uptake with carbonyl cyanidem -chlorophenylhydrazone (CCCP, 1μM) completely abolished cADPR-induced Ca2+signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca2+uptake pathways and that some of these pathways involve mitochondria.

The mouse 5-hydroxytryptamine 4(a) receptor [5-HT4(a)] was expressed with a baculovirus system in insect cells and analysed for acylation. [3H]Palmitic acid was effectively incorporated into 5-HT4(a) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Analysis of protein-bound fatty acids revealed that 5-HT4(a) contains predominantly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cells with BIMU8 {(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide}, a 5-HT4 receptor-selective agonist, generated a dose-dependent increase in [3H]palmitate incorporation into 5-HT4(a) with an EC50 of approx. 10nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects. We also used both pulse labelling and pulse–chase labelling to address the dynamics of 5-HT4(a) palmitoylation. Incorporation studies revealed that the rate of palmitate incorporation was increased approx. 3-fold after stimulation with agonist. Results of pulse–chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT4(a), thereby reducing the levels of receptor-bound palmitate to approximately one-half. Taken together, our results demonstrate that palmitoylation of 5-HT4(a) is a reversible process and that stimulation of 5-HT4(a) with agonist increases the turnover rate for receptor-bound palmitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylation in 5-hydroxytryptamine-mediated signalling.