Gymnodimine A and 13-desMethyl Spirolide C Alter Intracellular Calcium Levels via Acetylcholine Receptors.

Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]i). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca]i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca]i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca]i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.

Copper and cadmium administration induce toxicity and oxidative stress in the marine flatworm Macrostomum lignano.

The contamination of coastal regions with different toxicants, including heavy metal ions such as copper and cadmium jeopardize health and survival of organisms exposed to this habitat. To study the effects of high copper and cadmium concentrations in these marine environments, we used the flatworm Macrostomum lignano as a model. This platyhelminth lives in shallow coastal water and is exposed to high concentrations of all toxicants that accumulate in these sea floors. We could show that both, cadmium and copper show toxicity at higher concentrations, with copper being more toxic than cadmium. At concentrations below acute toxicity, a reduced long-term survival was observed for both metal ions. The effects of sublethal doses comprise reduced physical activities, an increase in ROS levels within the worms, and alterations of the mitochondrial biology. Moreover, cell death events were substantially increased in response to sublethal concentrations of both metal ions and stem cell activity was reduced following exposure to higher cadmium concentrations. Finally, the expression of several genes involved in xenobiotic metabolism was substantially altered by this intervention. Taken together, M. lignano has been identified as a suitable model for marine toxicological studies as it allows to quantify several relevant life-history traits as well as of physiological and behavioral read-outs.

Adjustments of molecular key components of branchial ion and pH regulation in Atlantic cod (Gadus morhua) in response to ocean acidification and warming.

Marine teleost fish sustain compensation of extracellular pH after exposure to hypercapnia by means of efficient ion and acid-base regulation. Elevated rates of ion and acid-base regulation under hypercapnia may be stimulated further by elevated temperature. Here, we characterized the regulation of transepithelial ion transporters (NKCC1, NBC1, SLC26A6, NHE1 and 2) and ATPases (Na(+)/K(+) ATPase and V-type H(+) ATPase) in gills of Atlantic cod (Gadus morhua) after 4 weeks of exposure to ambient and future PCO2 levels (550 µatm, 1200 µatm, 2200 µatm) at optimum (10 °C) and summer maximum temperature (18 °C), respectively. Gene expression of most branchial ion transporters revealed temperature- and dose-dependent responses to elevated PCO2. Transcriptional regulation resulted in stable protein expression at 10 °C, whereas expression of most transport proteins increased at medium PCO2 and 18 °C. mRNA and protein expression of distinct ion transport proteins were closely co-regulated, substantiating cellular functional relationships. Na(+)/K(+) ATPase capacities were PCO2 independent, but increased with acclimation temperature, whereas H(+) ATPase capacities were thermally compensated but decreased at medium PCO2 and 10 °C. When functional capacities of branchial ATPases were compared with mitochondrial F1Fo ATP-synthase strong correlations of F1Fo ATP-synthase and ATPase capacities generally indicate close coordination of branchial aerobic ATP demand and supply. Our data indicate physiological plasticity in the gills of cod to adjust to a warming, acidifying ocean within limits. In light of the interacting and non-linear, dose-dependent effects of both climate factors the role of these mechanisms in shaping resilience under climate change remains to be explored.

Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells.

To investigate the cellular accumulation and intracellular localization of dimercaptosuccinate-coated iron oxide nanoparticles (D-IONPs) in oligodendroglial cells, we have synthesized IONPs that contain the fluorescent dye BODIPY (BP) in their coat (BP-D-IONPs) and have investigated the potential effects of the absence or presence of this dye on the particle uptake by oligodendroglial OLN-93 cells. Fluorescent BP-D-IONPs and non-fluorescent D-IONPs had similar hydrodynamic diameters and ζ-potentials of around 60 nm and -58 mV, respectively, and showed identical colloidal stability in physiological media with increasing particle size and positivation of the ζ-potential in presence of serum. After exposure of oligodendroglial OLN-93 cells to BP-D-IONPs or D-IONPs in the absence of serum, the specific cellular iron content increased strongly to around 1,800 nmol/mg. This strong iron accumulation was lowered for both types of IONPs by around 50 % on exposure of the cells at 4 °C and by around 90 % on incubation in presence of 10 % serum. The accumulation of both D-IONPs and BP-D-IONPs in the absence of serum was not affected by endocytosis inhibitors, whereas in the presence of serum inhibitors of clathrin-dependent endocytosis lowered the particle accumulation by around 50 %. These data demonstrate that oligodendroglial cells efficiently accumulate IONPs by an endocytotic process which is strongly affected by the temperature and the presence of serum and that BP-D-IONPs are a reliable tool to monitor by fluorescence microscopy the uptake and cellular fate of D-IONPs.

Reporter dyes demonstrate functional expression of multidrug resistance proteins in the marine flatworm Macrostomum lignano: the sponge-derived dye Ageladine A is not a substrate of these transporters.

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells.

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ∼65 nm and -49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ∼130 nm and -8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3h with 150 and 450 µM iron as BP-IONPs increased the cellular iron content from a low basal level of ∼50 nmol iron mg(-1) to 219±52 and 481±28 nmol iron (mg protein)(-1), respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.

Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense.

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca(2+) influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca(2+) ions even during blockade of Ca(2+) channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.

Tracking of fast moving neuronal vesicles with ageladine A.

Ageladine A is a marine natural product that can be used to fluorescently stain living tissues and cells. Its fluorescence is highly pH dependent with the highest intensities under acidic conditions. We have used ageladine A to stain acidic vesicles in cells and found the compound especially useful for tracking transport vesicles in cultured nerve cells. Inward as well as outward ionic currents appear not to be influenced by ageladine A at concentrations of 10 µM or less. Higher concentrations than 30 µM reduce whole cell voltage dependent outward currents whereas inward currents remain unchanged up to 100 µM ageladine A (PC12 cells). Incubation with ageladine A (10 µM) in cultured hippocampal neurons does not alter miniature excitatory postsynaptic currents (mEPCS) amplitudes, frequency, rise or decay times. Fast moving vesicles are stained the brightest, suggesting they are the most acidic and likely to be Golgi derived and endocytotic vesicles for the fast anterograde and retrograde transport of proteins and other compounds needing an acidic environment.

Siphonodictyal B1 from a marine sponge increases intracellular calcium levels comparable to the Ca2+-ATPase (SERCA) inhibitor thapsigargin.

Siphonodictyal B1 is a sesquiterpene-hydroquinone isolated from the Caribbean coral reef bioeroding sponge Siphonodictyon coralliphagum. Siphonodictyal B1 increased intracellular calcium levels in neuroendocrine cells (PC12) in the presence and absence of extracellular calcium using Fura-2 as a calcium-sensitive dye. The calcium rise was comparable in amplitude and timing to the application of the sarco-endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin from the terrestrial plant Thapsia garganica. The effects of thapsigargin and siphonodictyal B1 on intracellular calcium levels were not distinguishable in pharmacological experiments conducted with caffeine, ryanodine, muscarine, and thapsigargin in calcium-free and calcium-containing buffer, although thapsigargin was effective at lower concentrations. Thapsigargin is a sesquiterpene-lactone and has no structural similarities to siphonodictyal B1. We conclude that thapsigargin and siphonodictyal B1 share SERCAs as cellular targets. Siphonodictyal B1 may be involved in the process of bioeroding the calcium carbonate endoskeleton of the scleractinian corals attacked by S. coralliphagum.

Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye.

The alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.

Disturbance of voltage-induced cellular calcium entry by marine dimeric and tetrameric pyrrole--imidazole alkaloids.

Twelve brominated pyrrole-imidazole alkaloids from the Caribbean sponges Stylissa caribica and Agelas wiedenmayeri were tested for interactions with cellular calcium homeostasis using PC12 cells. Massadine (half maximal concentration: 5.32 +/- 0.007microM), stylissadines A (4.48 +/- 1.1microM) and B (4.6 +/- 1.6microM) as well as tetrabromostyloguanidine (15.6 +/- 0.004microM) reduced voltage-dependent calcium entry in PC12 cells as measured with Fura II as calcium indicator. Dibromopalau'amine and mauritiamine reduced voltage-dependent calcium entry but no half maximal concentration can be calculated from our results. Monomeric brominated pyrrole alkaloids such as stevensine, cyclooroidin, oxocyclostylidol, 4-bromopyrrole-2-carboxy-N(epsilon)-lysine, and 4-bromopyrrole-2-carboxyarginine showed no or only minor effects. Ageladine A itself showed fluorescence in a similar range as Fura II and therefore no data are reported here. Based on the results a structure-activity relationship could be established. Absolutely necessary for an activity seem to be a lipophilic (brominated side chain) and a hydrophilic (amino-imidazole core) substructure. The combination of these substructures may be on one hand responsible for the membrane solubility (dibromopyrrole moieties) and on the other hand for the interaction with the hydrophilic area of the calcium channel (amino-imidazole moieties) to accomplish the alkaloids neurotoxic potential.

The marine secondary metabolites 2,4-dibromophenol and 2,4,6-tribromophenol differentially modulate voltage dependent ion currents in neuroendocrine (PC12) cells.

2,4-Dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) are marine secondary metabolites, with 2,4,6-tribromophenol playing an important role as industrially produced flame retardant and pesticide. Both substances disturb cellular calcium signals in neuroendocrine cells as previously shown by Hassenklöver et al. (2006) [Hassenklöver, T., Predehl, S., Pilli, J., Ledwolorz, J., Assmann, M., Bickmeyer, U., 2006. Bromophenols, both present in marine organisms and in industrial flame retardants, disturb cellular Ca(2+) signaling in neuroendocrine cells (PC12). Aquat. Toxicol. 76, 37-45]. We investigated calcium channel currents in detail and outward membrane currents as potential cellular targets of both bromophenols. In this electrophysiological approach, 2,4-DBP reduced voltage dependent calcium channel currents with a half-maximal concentration of 45+/-32 microM (S.D.) and a Hill coefficient of 0.87+/-0.49 (S.D.). 2,4,6-TBP reduced calcium channel currents with a half-maximal concentration of 28+/-19 microM (S.D.) and a Hill coefficient of 0.79+/-0.31 (S.D.). The major contribution to calcium channel currents was mediated by L-type (67%) and N-type channels (30%) in PC12 cells; both bromophenols modulated both current types. Whole cell outward currents, mainly carried by potassium ions, were reduced by 2,4-DBP with a half-maximal concentration of 41+/-9 microM (S.D.) showing a Hill coefficient of 1.71+/-0.31 (S.D.). 2,4,6-TBP showed a weak reduction of outward currents at high concentrations of 300 microM. 2,4,6-TBP selectively decreased calcium entry via calcium channels as revealed in whole cell patch clamp experiments, whereas 2,4-DBP reduced both in- and outward currents.

Bromophenols, both present in marine organisms and in industrial flame retardants, disturb cellular Ca2+ signaling in neuroendocrine cells (PC12).

Bromophenols are present in polychaetes as well as in algae in marine environments including the North Sea. They are thought to cause the typical sea-like taste and flavour. The ecological function of brominated phenols is not clear yet, but they may play a role in chemical defence and deterrence [Kicklighter, C.E., Kubaneck, J., Hay, M.E., 2004. Do brominated natural products defend marine worms from consumers? Some do, most don't. Limnol. Oceanogr. 49, 430-441]. Some brominated phenols are commercially used as industrial flame retardants as, e.g., 2,4,6-tribromophenol and are suspected to disrupt the humoral system by showing thyroid hormone-like activity [Legler, I., Brouwer, A., 2003. Are brominated flame retardants endocrine disruptors? Environ. Int. 29, 879-885]. In this study 2-bromophenol (2-BP), 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,6-dibromophenol (2,6-DBP) and 2,4,6-tribromophenol (2,4,6-TBP), all of which are present in marine organisms, were tested. Especially 2,4-DBP and 2,4,6-TBP showed a significant effect on the Ca2+ homeostasis in endocrine cells (PC 12). The reduction of depolarization induced Ca2+ elevations by 2,4-DBP and 2,4,6-TBP and the increase of intracellular Ca2+ by both substances, partly released from intracellular stores, may suggest a link to the disrupting effect of endocrine systems by brominated phenols. 2,4-DBP was the most potent substance we tested in respect to inhibition of voltage dependent Ca2+ currents as revealed in whole cell patch clamp experiments. Brominated phenols disturb cellular Ca2+ signaling with differential efficacy, depending on the number and position of bromine.

Bromoageliferin and dibromoageliferin, secondary metabolites from the marine sponge Agelas conifera, inhibit voltage-operated, but not store-operated calcium entry in PC12 cells.

Two alkaloids isolated from the marine sponge Agelas conifera were tested for interactions with cellular calcium homeostasis. Bromoageliferin and dibromoageliferin reduced voltage-dependent calcium entry in PC12 cells as measured with Fura II as calcium indicator. The half maximal concentration of both alkaloids to reduce voltage-dependent calcium entry was only slightly different: bromoageliferin showed a half maximal concentration of 6.61+/-0.33 microM, dibromoageliferin of 4.44+/-0.59 microM. Removal of calcium from extracellular solution for 10 min leads to an, at least, partial depletion of intracellular calcium stores, which induces a store-operated calcium entry after re-supplementation of calcium to the buffer. The store-operated calcium entry was unchanged by dibromoageliferin at a concentration of 30 microM, which fully blocks voltage-dependent calcium entry. The store-operated calcium entry induced by application of 5 microM thapsigargin was similarly not altered by 30 microM bromoageliferin. Both alkaloids reduce voltage-dependent calcium entry, but not store-operated calcium entry. The inhibition of voltage-operated calcium entry by bromoageliferin is shown in whole-cell patch clamp experiments.

Brominated pyrrole alkaloids from marine Agelas sponges reduce depolarization-induced cellular calcium elevation.

Seven pyrrole alkaloids isolated from Agelas sponges were tested for interactions with the cellular calcium homeostasis. Brominated pyrrole alkaloids reduced voltage dependent calcium elevation in PC12 cells. Dibromosceptrin was the most potent alkaloid with a half maximal concentration of 2.8 microM followed by sceptrin (67.5 microM) and oroidin (75.8 microM). 4,5-Dibromopyrrole-2-carboxylic acid reduced calcium elevation at concentrations exceeding 30 microM but did not eliminate calcium elevation at concentrations up to 1 mM. 4-Bromopyrrole-2-carboxylic acid and pyrrole-2-carboxylic acid were not active in this respect. The aminoimidazole group appeared to have a significant effect on voltage dependent calcium elevation shown by the comparison of oroidin with 4,5-dibromopyrrole-2-carboxylic acid. The degree of bromination of the pyrrole moiety is another important factor, as was shown by the comparison of 4,5-dibromopyrrole-2-carboxylic acid with 4-bromopyrrole-2-carboxylic acid, as well as oroidin with hymenidin and dibromosceptrin with sceptrin. The previously reported feeding deterrent activity of brominated pyrrole alkaloids in Agelas sponges against predatory reef fish may partly be explained by a general interaction of these alkaloids with the cellular calcium homeostasis. The chemoreception of bromopyrrole alkaloids in sea water is shown using sensory neurons in the rhinophore of the sea slug Aplysia punctata.

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel.

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.

The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

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.