Roman Chaloupka

New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.

pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pHin) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pHin for three prototrophic wild‐types. The new construct was also used to determine the pHin in strains differing in the activity of the plasma membrane Pma1 H+‐ATPase and the influence of glucose on pHin. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands‐on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright

Hypocrellin A Photosensitization Involves an Intracellular pH Decrease in 3T3 Cells

Abstract— The fluorescent pH probe carboxy‐seminaphtorhoda‐fluor‐1 (C‐Snarf‐1) has been used for laser microspectrofluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al, J. Am. Chem. Soc. 118, 9484‐9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 μM hypocrellin A after 90 s of exposure to 0.1 μW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A‐treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C‐Snarf‐1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C‐Snarf‐1 that results in an increase of C‐Snarf‐1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

Over-expression of Bcl-2 does not protect cells from hypericin photo-induced mitochondrial membrane depolarization, but delays subsequent events in the apoptotic pathway.

Hypericin (HY) is a powerful photo‐inducer of apoptosis in Jurkat cells as measured by caspase‐3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl‐2‐independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre‐incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (Δψ m) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo‐induced mitochondrial permeability. This should be considered as the result of a non‐specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti‐apoptotic protein Bcl‐2 appears to delay the subsequent time course of PS exposure and to reduce caspase‐3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl‐2 with cytosolic cytochrome c previously released from mitochondria upon Δψ m decrease and/or of Bcl‐2 inhibition of the deleterious retro‐effect of caspase‐3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6

Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me2+ and H+ into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd2+ and H+ uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp34 couples H+ and Me2+ symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His211 residue mediates pH-dependent Me2+ uptake; MntH Asn37, Asn250, and Asn401 in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp34-Pro-Gly (TMS1) and Met-Pro-His211 (TMS6) form antiparallel “TM helix/extended peptide” boundaries, lining a “pore” cavity and enabling H+-dependent Me2+ import.

Monitoring the kinetics and performance of yeast membrane ABC transporters by diS-C3(3) fluorescence.

Kinetic features (initial start-up phase, drug pumping velocity and efficiency as dependent on drug concentration and growth phase) of yeast plasma membrane multidrug resistance ABC pumps were studied by monitoring the uptake of the fluorescent potentiometric dye diS-C3(3), which has been found to be expelled from the cells by these pumps. The monitoring was done with Saccharomyces cerevisiae mutants AD1-8 and AD1-3 deleted in different ABC pumps, and in their pump-competent parent strain US50-18C overexpressing transcriptional activators Pdr1p and Pdr3p. On addition to the cells, diS-C3(3) is expelled by the Pdr5p, Yor1p and Snq2p pumps with overlapping substrate specificity. The pump action can be assessed as a difference between the dye uptake curve for pump-competent and pump-deleted cells. The pump-mediated dye efflux, which shows an initial lag of various lengths, maintains a certain residual intracellular dye level. In the absence of external glucose the dye efflux ability of the pumps depends on the growth phase; late exponential and stationary cells can maintain the export for tens of minutes, whereas exponential cells keep up the pump action for limited time periods. This may reflect an insufficient number of pump molecules in the membrane or an effect of insufficient pump energization from endogenous sources. This effect is not mediated by changes in membrane potential because lowered membrane potential caused by inhibition of the plasma membrane H+-ATPase does not affect the pump action.

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast

The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max). These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.

Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure-function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His(211) were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure-function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein.