Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.

Pressure-flow curves for control and hardened (diamide treated) human RBC's were obtained in capillaries of the isolated rat mesentery, in order to evaluate resistance to flow of hardened RBC's. Blood vessels were maximally dilated by an infusion of 10(-5) mol/l acetylcholine and isoprenaline and perfused with freshly collected human RBC's as well as with RBC's hardened by a treatment (hct 40%; pH 8.0; 37 degree C) with 0.5 mmol/l or 1.5 mmol/l diamide, respectively, suspended in Albumin (0.05%) - Ringer solution. The mesentery was perfused via a hydrostatic pressure reservoir. Arterio-venous pressure difference was varied from 4-10 kPa, and corresponding arteriolo-venular pressure gradients changed from about 200-500 Pa/mm. No significant difference in resistance to flow was observed between control and diamide treated cells over the whole pressure range. However, the flow through the microvascular bed was inhomogeneous upon perfusion with diamide treated cells, caused by a deceleration and stoppage of the cells at capillary narrowing (ratio of cell to vessel diameter greater than 2). The time of stagnation increased with decreasing pressure gradient.

A graded reduction of "deformability" of red blood cells (RBC's) of rats was obtained by treatment with the SH-oxidizing agent, diamide. Rigidified RBC's were injected into rats by isovolemic exchange against 60% of the native RBC's and RBC flow velocities in capillaries of rat mesentery measured. At normal mean arterial pressure RBC flow velocity decreases by 29% in rats receiving cells rigidified with 0.5 mmol . 1(-1) diamide. Surprisingly a further rigidification of erythrocytes by 1.5 mmol . 1(-1) diamide results in a decrease of flow by only 15%. During hypotension RBC flow velocities dropped precipitously to 8 +/- 15% for the 0.5 nmol . 1(-1) and to 2 +/- 6% for the 1.5 mmol . 1(-1) diamide group compared to velocities during normotension. By microscopy we observed a stop of flow in many vessels. This result outlines the importance of a normal red cell "deformability" for the maintenance of sufficient perfusion of the microcirculation, in particular at low blood pressure gradients.