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Research topics

Projects 1.  Regulation of sodium-dependent solute transport by physico-chemical parameters 2.  Magnesium transport      -  Transmembranal Magnesium transport      -  Transepithelial Magnesium transport   Methods -  Transport on isolated brush border membrane vesicles (BBMV) -  Determination of transport on intact erythrocytes   Publications

Projects

1. Regulation of Na⁺ dependent solute transport by physico-chemical parameters

It is aimed to investigate how Na⁺-dependent solute transport is regulated by membrane fluidity, membrane surface charge, membrane thickness and other physico-chemical parameters. Previously we showed that Na⁺-dependent D-glucose transport into brush border membrane vesicles (BBMV) of kidney and intestine is closely related to membrane fluidity: an increase in membrane fluidity as produced by incorporation of long chain unsaturated fatty acids (LCUFA) led to a decrease of Na⁺-dependent D-glucose transport which was obviously mediated by a conformational change of the transport protein. In addition, also trans Na⁺ inhibition contributes to transport inhibition.

Current investigations are concerned with two topics:

• Specificity of the LCUFA effect: the effect of LCUFA on other Na⁺-dependent and Na⁺-independent transporter for sugars, carboxylic acids and amino acids will be investigated. The results may have clinical relevance for slowing intestinal glucose absorption in diabetics or obese patients by increased intake of LCUFA.
• Role of membrane surface charge: membrane surface charge will be varied by incorporation of phospholipids into BBMV that have a neutral, positive or negative head group charge and are designed with different acyl chains (saturated versus unsaturated with different chain length).

2. Mg²⁺ transport

Transmembranal Mg²⁺ transport

Since most cells exhibit a relatively low Mg²⁺ exchange, it has been difficult to investigate Mg²⁺ transport so that not to much is known on its mechanism. To obtain measurable Mg²⁺ efflux rates in human erythrocytes (as a model for transmembrane Mg²⁺ transport) it has been necessary to load these cells massively with Mg²⁺. Under this rather un-physiological condition it has been found that Mg²⁺ is only partially transported by a Na⁺/Mg²⁺ antiport. The mechanism of the Na⁺ independent Mg²⁺ efflux remained unclear.

We want to characterize the mechanism of Mg²⁺ efflux under more physiological conditions in non Mg²⁺-loaded erythrocytes. This will be done on rat erythrocytes that have approximately a 50 times higher Mg²⁺ efflux than human erythrocytes so that Mg²⁺ loading is not necessary, which may change the properties of the Mg²⁺ transporter by an allosterically induced conformational change.

Transepithelial Mg²⁺ transport

In absorbing epithelia like kidney and intestine Mg²⁺ is taken up through the tight junctions but also by a membranal transport process. In the kidney there is cumulative evidence for transmembranal electrogenic and Na⁺ independent Mg²⁺ transport through a channel. In the intestine it is debated whether there is a transmembranal Mg²⁺ transport. Recently we showed that BBMV from the rabbit ileum exhibit a saturable, electroneutral and anion sensitive Mg²⁺ transport. It remained unclear whether this Mg²⁺ transport is performed by a transport protein or by a channel and what is the role of the anions.

Aim of our continued investigations is to characterize in detail the role of anions and protons on Mg²⁺ transport in BBMV from the rabbit ileum. To do this, it is necessary to determine free Mg²⁺ with the fluorophore Mag-Fura 2 in the BBMV, since it tends to bind strongly to the membranes thus mimicking false transport. This method which usually is applied to intact cells has been already established by us in isolated BBMV.

Methods

• Isolation of BBMV by MgCl₂ precipitation and subsequent differential centrifugation
• Determination of Na⁺ dependent solute transport with ³H and/or ¹⁴C labeled solutes and the rapid membrane filtration method
• Determination of Mg²⁺ transport by fluorometric determination of intravesicular free Mg²⁺ by the Mag-Fura 2 method (Two wavelengths method)
• Variation of cation, anion and proton gradients directed from outside to inside or inside to outside
• Variation of membrane surface charge, membrane fluidity and membrane thickness by incorporation of phospholipids with a different headgroup charge (neutral, positive and negative) and differently saturated acyl chains of different chain length
• Determination of membrane "fluidity" by measuring fluorescence depolarization with probes for different membrane regions (DPH, 12-AS).
• Determination of membrane surface potential by merocyanin and the DiSC₃ (5) fluorescence method
• Determination of intravesicular pH with the BCECEF and SNARF fluorescence methods

Determination of transport on intact erythrocytes

• Use of erythrocytes of different species
• Determination of Mg²⁺ efflux in to Mg²⁺-free medium on non Mg²⁺-loaded and Mg²⁺-loaded erythrocytes
• Differentiation of Na⁺-dependent and Na⁺-independent Mg²⁺ efflux in different media of low and high ionic strength
• Variation of extracellular cation, anion and proton gradients and of the membrane potential after Mg²⁺ depletion and Mg²⁺ loading

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