Prof. Alide Faelli, Prof. Giuliano Meyer, Prof. Maria Novella Orsenigo, Dr. Guido Bottà.

The research lines run by this Unit include the molecular physiology and the physiology of membrane transports. Our scientific interest area in molecular physiology concernes the structure-function correlation and protein-protein interaction study of different membrane proteins. In particular we are interested in the involvement of the ICln protein, an ion channel cloned from a dog renal epithelial cell line, in cell volume regulation. In the field of transport physiology we are studying the function and the molecular identification of membrane carriers and channels.  We are analysing the membrane transport regulations as a function of the cellular homeostasis and of the transepithelial absorptions or secretions. Our laboratory team has a long experience in  electrophysiological, radiochemical, fluorimetric and molecular biology techniques.

Research topics:

Molecular Physiology:

1. Structural and functional characterization of the ICln channel by site-directed deletions and reconstitution in lipid bilayer

Paulmichl, Meyer, Bottà, Dopinto, Salvioni.

ICln is a cytoplasmic water-soluble protein, which can be incorporated into the cellular membrane, where it acts as an  ion channel. The channel mediates a chloride current resembling that activated after cell swelling (ICl_swell ), which allows the cells to perform regulatory volume decrease (RVD). Recently, we have identified as a PH-domain the structure of the ICln protein in its water soluble form. This finding, together with other functions linked to the methylosome, enhances the role of Icln as an hub protein according to a cellular interactome network description. Our present aim is the characterization of the biophysical features elicited by the ICln channel reconstituted in artificial lipid bilayers made of sphingomyeline mixtures. In particular, we search for functional variations between ICln-wild type and different isoforms obtained with site-directed deletions in order to understand the role of specific amino acids on the channel activity.

2. Functional reconstitution of ICln channel in raft-like membranes: influence of cholesterol concentration and localization in the lipid rafts

 Paulmichl, Meyer, Bottà, Dopinto, Bazzini.

Lipid rafts are dynamic assemblies of proteins and lipids, enriched with sphingolipids and cholesterol, moving freely within the liquid bilayer. Cholesterol has been identified as a key factor in determining lipid rafts stability and organization, and it has been shown to be an important component of raft-related structures as well (e.g. caveolae). Studies using cholesterol-containing bilayers as model systems have provided important insights into the role played by cholesterol. High cholesterol is able to disrupt rafts formation therefore we set out to investigate the role of cholesterol in bilayers with sphingomyelin-rich domains. We are also investigating the subcellular localization of ICln channels expressed in NIH3T3 cells. Our approach involves different methods based on: 1) FRET (Fluorescence Resonance Energy Transfer; 2) discontinuous sucrose gradient followed by western blot analysis to detect the low-density membrane fractions that co-localise  with flotillin or caveolin as thypical markers of rafts or caveole.

Fig. 1. NMR-derived structure of water-soluble ICln159.

a, a bundle of 15 final structures and a ribbon plot of ICln with the loop between β6/β7 displayed in magenta, β-sheets are in cyan, and the α-helix is in red. b, T2 versus amino acid sequence of the backbone amide protons. The loop between strand β6 and β7 is disordered and highly mobile and indicated with a bar in magenta. Green columns indicate the β-sheets, and the gray column indicates the  α-helix.

3. The PH domain of ICln and the interaction with phosphatidylinositols

Paulmichl, Meyer, Bottà, Bazzini.

In a variety of cell types, swelling coincides with an increase in phosphatidylinositol-3-kinase (PI-3-K) activity. In particular, with patch-clamp experiments, we saw that pre-treatment of the cells with inhibitors of PI-3-K inhibits the volume-sensitive ion efflux. With parallel structural studies (NMR, performed in collaboration with Prof. Konrat, Costanza.) we demonstrated that water soluble ICln is a PH domain (fig 1). This domain is usually involved in protein-protein interactions or phosphatidylinositols (PtdIns) binding, suggesting a possible role of PtdIns in the traslocation of ICln to the membrane and/or the activation of the ICl,swell current.
On these basis we are currently studying the interaction between ICln and different PtdIns, especially 3-phosphoinositols, by in vitro techniques (protein overlay experiments, liposome binding experiments) and in vivo experiments (patch-clamp, FRET).

4. ICln interacting proteins

Paulmichl, Meyer, Bottà, Dossena, Vezzoli, Bazzini.

Beside its role in cell volume regulation, additional functions of ICln in other regulatory modules, i.e. the regulation of cell morphology, platelet activation or RNA processing, have been described. The finding that ICln interacts with cytosolic and membrane or membrane associated proteins, suggests that it may function as a connector hub linking various regulatory pathways and compartments within a cell. As a step towards a better understanding of the function of ICln, we are trying to identify the ICln patners in the cytosolic fraction as well as in the membrane fraction and we are mapping the proteome changes in cells during the regulatory volume decrease.
Particularly, we mean to investigate the functional and molecular interaction between ICln and the platelet integrin alpbaIIb chain, as well as a putative zink finger protein of unknown function termed HSPC038. These experiments could help to elucidate the mechanisms leading to the transposition of ICln towards the cell membrane during the regulatory volume decrease, as well as the role of ICln and the interacting proteins in the activation of the anion current elicited by cell swelling (ICl,swell).

5. ICln and apoptosis

Paulmichl, Meyer, Bottà, Bazzini.

Cell proliferation and apoptosis are basic biological processes through which new cells are formed, and damaged cells are eliminated to ensure body growth and organ repair. Apoptosis is a complex process, involving several steps that finally lead to cell death. An early key event is the activation of an anion current that closely resembles the swelling activated chloride current (ICl,swell). The activation of the current causes an efflux of ions and consequent cell shrinkage, known as Apoptotic Volume Decrease (AVD, Fig 2). Since ICln is essential for the activation of ICl,_swell, we are investigating the relationship between ICln and the AVD current, apoptosis and cell proliferation. The project involves the use of vitality assays and measures of the activation of key events of the apoptotic pathway, performed on cells over-expressing ICln or after silencing of ICln by RNAi.

6. Transcriptional regulation of the ICln gene

Paulmichl, Meyer, Bottà, Vezzoli.

By bioinformatic analysis, we have found in the 5’ flanking region of the ICln gene a sequence element (IRES) common in those genes that are involved in important physiological process like apoptosis and response to the cellular stresses. Consequently we are studying if this element could regulate ICln expression during the regulatory volume decrease.

Fig. 2 Ionic mechanisms of volume regulation and cell death induction

Transport Physiology:

1. Essential hypertension: functional and molecular alteration of distal nephron ion channels

Meyer, Bottà, Paulmichl, Garavaglia, Bononi, Saino.

Essential hypertension is closely associated with an increase in  renal Na+ reabsorption. This leads to hypervolemia, which is a crucial step in the development of hypertension.
The distal tracts of the nephron (thick ascending limb, distal convoluted tubule and collecting duct) are the regions where NaCl is actively absorbed, which induces a regulated water reabsorption as a result of the generated osmotic gradient. Since the ion channels both in the apical and basolateral membrane are functionally correlated with the electroneutral transports for NaCl reabsorption in distal nephron, cotransporters modifications could be associated to an altered expression and/or function of ion channels. We are studying  the possible functional alterations of these ion channels in Milano hypertensive rats (MHS), a system model of human essential hypertension. 

2. Effect of S-Carbocysteine-lysine salt monohydrate on glutathione secretion from respiratory epithelium

Meyer, Bottà, Paulmichl, Garavaglia, Bononi, Guizzardi, Rodighiero.

Glutathione (GSH) is one of the major antioxidative agents in the surface fluids covering the higher airways mucosae. GSH can be secreted in an electrogenic way which is dependent on CFTR (Cystic Fibrosis Trasmembrane Regulator) anion channel. This Research Unit studies, by using electrophysiological techniques, the ability of S-Carbocysteine-lysine salt monohydrate (a promoter of mucolysis) to stimulate both Cl- and GSH secretion in human respiratory epithelium cells. Effects of S-Carbocysteine-lysine salt monohydrate also in the presence of oxidative stress will be analysed. 

3. Functional and molecular characterization of human pendrin

Bottà, Paulmichl, Meyer, Dossena, Vezzoli.

Pendrin is a recently cloned membrane anion exchanger; it is mainly expressed in the thyroid, in the inner ear and in the kidney. It plays a fundamental role for a correct inner ear development and for the thyroid hormones biosynthesis. Mutations of the protein lead to Pendred’s syndrome, an autosomal recessive disease characterized by sensorineural hearing loss and a partial iodide organification defect that could lead to goitre development and hypothyroidism.
We are studying the transcriptional mechanisms leading to Pendrin gene expression, characterizing the promoter region in different cellular lines. Moreover, we are conducting experiments (western blot analysis, immunohistochemistry, transport analysis with fluorescent and radioactive markers, etc) in order to molecularly and functionally characterize the wild type Pendrin and some natural occurring mutants identified in patients with Pendred syndrome. These studies are aimed at individuating  mechanisms (as chaperon proteins, drugs, physical conditions) that could possibly restore the function of the defective proteins.

Fig. 3 Expression of exogenous Pendrin in HEK293-Phoenix cells. The subcellular localization was done by co-expressing Pendrin (red signal) together with a EYFP conjugated plasma membrane label (green signal).

4. Creatine transport in the jejunal tract of intestine

Faelli, Orsenigo, Tosco.

Creatine dietary supplement is increasingly used both as an ergogenic aid to improve athletes exercise performance and as a possible therapeutic agent in the treatment of various muscle, neurological and neuromuscolar diseases. In spite of the physiological relevance of oral creatine intake, up to now little is known about the mechanisms of intestinal creatine absorption.  

5. Endogenous transport mechanisms for monocarboxylates in the plasma membrane of Xenopus laevis oocyte and their regulation

Orsenigo, Tosco, Faelli.

Xenopus laevis oocytes are glycogenic cells that use pyruvate and lactate as main carbon source. Pyruvate and lactate derive in a small part from glycolysis, but mostly they are introduced into the oocyte by means of a Na⁺-dependent transport mechanism located on its plasma membrane. Aim of this research is to investigate the possible effects of various protein kinases activation on the function of the endogenous monocarboxylate transporter in Xenopus laevis oocyte. This modulation could play a physiological role in the deep metabolic changes that are involved in oocyte maturation.

6. Effect of oxidative stress on intestinal transporters

Tosco, Orsenigo, Faelli.

Various oxidative stress parameters have been studied in the gut, since intestine is exposed to oxidant agents of both exogenous (diet and lumen) and endogenous (mucosa) origin. Aim of this research is to investigate the effect of different oxidant agents (H₂O₂, O₂^·-, ^·OH)  on the activity of rat jejunal (Na⁺, K⁺)-ATPase, responsible for transepithelial Na⁺ transport. Moreover we intend to examine the possible direct effects of the same agents on single transport mechanisms involved in the transepithelial transports of fluid, glucose, sodium, chloride and lactate, located both in the apical and in the basolateral membrane of enterocyte.

Publications

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