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Differential association of psychopharmacological compounds with membrane microdomains in relation to the modulation of ligand-gated ion channels

The concept of lipid rafts has attracted considerable interest, because these membrane domains have been implicated in numerous cellular functions such as membrane trafficking and signal transduction processes. We have shown that antidepressants and antipsychotics colocalize with 5-HT3 receptors in raft-like domains and that they inhibit serotonin-evoked cation currents through 5-HT3 receptors in a noncompetitive manner (Eisensamer et al. 2005). However, there is increasing evidence that the raft association of distinct proteins largely depends on the biochemical preparation procedure (Nothdurfter et al. 2007) (Fig. 1). Using a detergent-free protocol, the 5-HT3 receptor is predominantly found in raft-like domains as shown by means of Western blot analysis (Fig. 2).

 

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Fig.1: Separation of the raft marker protein caveolin-1 and the non-raft protein transferring receptor by the detergent Triton X-100.

 

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Fig. 2: Distribution of the 5-HT3 receptor protein in membrane fractions of sucrose density gradients

 

In contrast to sucrose density gradients, immunocytochemistry revealed only a weak colocalization between the 5-HT3 receptor and raft marker proteins (Fig. 3). Moreover, cholesterol depletion by ß-methylcyclodextrin showed an impairment of lipid raft integrity in immunocytochemistry but did not prevent the effects of DMI on 5-HT3 receptor function (Fig. 4) thereby indicating that the allosteric modulation of 5-HT3 receptors by antidepressants involves also non-raft 5-HT3 receptors. In conclusion, the differential accumulation of psychopharmacological compounds in relation to their colocalization with neurotransmitter subunits within the cell membrane may represent an important determinant for their effects on neuronal excitability, which may play a role for their distinct pharmacological profile both with regard to clinical effects and side effects.

 

 

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Fig. 3: Colocalization of the 5-HT3 receptor and flotillin-1 in N1E-115 cells A Immunofluorescence of the 5-HT3 receptor and flotillin-1 B Magnification of merge from A. and Intensity profiles of the red (flotillin-1) and the green ( 5-HT3 receptor) channels.


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Fig. 3: Impact of cholesterol depletion by means of methyl-ß-cyclodextrin (MßCD) on 5-HT evoked cation currents and its modulation by the antidepressant desipramine (DMI) in N1E-115 cells


Research groups involved:

linkWebsiteRG Rainer Rupprecht: Caroline Nothdurfter, Sascha Tanasic, Eva-Maria Wagner, Barbara Di Benedetto, Julia Kessler

linkWebsiteRG Theo Rein: Thomas Kirmeier

linkWebsiteRG Manfred Uhr

linkWebsiteRG Matthias Eder: Gerhard Rammes

linkWebsiteGerald Gimpl, Insitute of Biochemistry, Johannes Gutenberg University Mainz

linkWebsiteJeremy Lambert, Neuroscience Institute, Division of Pathology & Neuroscience, Ninewells Hospital & Medical School, Dundee University

 

Publications:

Nothdurfter C, Tanasic, S, Rammes G, Rupprecht R (2011) Modulation of ligand gated ion channels as a novel pharmacological principle. Pharmacopsychiatry 44: 527-534

 

Nothdurfter C, Tanasic, S, di Benedetto B, Rammes G, Wagner EM, Kirmeier T, Ganal V, Kessler JS, Rein T, Holsboer F, Rupprecht R (2010) Impact of lipid raft integrity on 5-HT3 receptor function and its modulation by antidepressants. Neuropsychopharmacology 35: 1510-1519

 

Nothdurfter C, Rammes G, Rein T, Rupprecht R: (2007) Pitfalls in isolating lipid rafts. Nature Reviews Neuroscience 8: 567

 

Eisensamer B, Uhr M, Meyr S, Gimpl G, Deiml T, Rammes G, Lambert J, Zieglgänsberger W, Holsboer F, Rupprecht R (2005) Antidepressants and antipsychotic drugs co-localize with serotonin type 3 (5-HT3) receptors in raft-like domains. Journal of Neuroscience 25: 10198-10206