Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca 2؉ flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1␣, RANTES (regulated on activation normal T cell expressed), and MIP-1␤). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wildtype cells in order to produce the same intracytoplasmic Ca 2؉ flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1␣ than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1␤. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1␣, RANTES, or MIP-1␤. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.

1. Whole-cell patch clamp recordings were used to investigate the modulation by reducing and oxidizing agents of recombinant human cardiac L-type Ca¥ channel á1C subunits stably expressed in human embryonic kidney (HEK 293) cells. 2. The oxidizing agents thimerosal (10 ìÒ) and p-chloromercuribenzene sulphonic acid (PCMBS; 2 ìÒ to 2 mÒ) caused irreversible inhibition of Ca¥ channel currents. The reducing agent 1,4-dithiothreitol (DTT; 2 mÒ) was without effect on Ca¥ channel currents, but reversed the inhibitory actions of thimerosal and PCMBS. 3. Ca¥ channel currents were also inhibited by pretreatment with the methanethiosulphonate compound (2-aminoethyl)methanethiosulphonate (MTSEA, 2·5 mÒ), but were unaffected by identical pretreatment with (2-sulphonatoethyl)methanethiosulphonate (MTSES, 10 mÒ). The effects of MTSEA could be fully reversed by DTT (2 mÒ). The degree of current inhibition caused by 200 ìÒ PCMBS was not significantly affected by pretreatment with MTSEA, and following PCMBS treatment, MTSEA caused a similar degree of inhibition to that observed in cells that were not previously treated with PCMBS. These findings suggested that distinct thiol groups were modulated by these two agents. 4. Hypoxic inhibition of Ca¥ channel currents was unaffected by pretreatment of cells with MTSEA but was fully prevented by treatment with PCMBS. Our results indicate that distinct cysteine residues on the á1C subunit can undergo redox modulation and in so doing alter channel function. Some, but not all, of these residues appear to be associated with the mechanism underlying inhibition of this channel by hypoxia.

1. Whole-cell patch clamp recordings were used to investigate the effects of hypoxia on recombinant human L-type Ca2P channel a1c subunits stably expressed in human embryonic kidney (HEK 293) cells.2. Ca2P channel currents were reversibly inhibited by hypoxia (Po2 < 90 mmHg). The degree of inhibition depended on the charge carrier used, Ca2+ currents being more 02 sensitive than Ba2+ currents. 3. Hypoxic inhibition of Ca2+ channel currents was more pronounced at lower activating membrane potentials (< +30 mV), and was associated with a slowing of activation kinetics. Current inactivation and deactivation were unaffected by hypoxia. 4. Since hypoxia similarly regulates native L-type Ca2+ channels in vascular smooth muscle cells, our results suggest that hypoxic regulation of L-type Ca2+ channels arises from modification of structural features of the a1 subunit common to cardiac and smooth muscle L-type channels.

Chemokines interact with specific G-protein-coupled cell-surface receptors and with glycosaminoglycans (GAGs), such as heparan sulphate. Although chemokines often form multimers in solution, this process may be enhanced following interaction with GAGs on the cell surface, or within the extracellular matrix. However, the significance of multimerization for chemokine function remains controversial. In the present study, a fusion protein was prepared between the prototypical human CC chemokine, monocyte chemoattractant protein-1 (MCP-1; also known as CCL-2) and a large secreted placental alkaline phosphatase (SEAP) moiety. This fusion protein (MCP-1–SEAP) remained monomeric under conditions that promote oligomerization of the native chemokine. Radioligand binding showed that both native MCP-1 and MCP-1–SEAP competed for the same site on the surface of HEK-293 cells expressing the CCR2b chemokine receptor. The interaction between either chemokine species and endothelial cell surface GAGs was antagonized by the addition of the heparan sulphate-like molecule, heparin. Both MCP-1 and MCP-1–SEAP induced a Ca2+-flux in the THP-1 monocytic cell line, and were equally effective at promoting transendothelial chemotaxis of mononuclear immune cells, with maximal migration being produced by treatment with 12nM of either species. In each case this chemotactic response was almost completely antagonized by the addition of heparin. The importance of interaction between either native MCP-1 or MCP-1–SEAP and cell-surface GAGs for transcellular migration was demonstrated by the almost complete absence of leucocyte chemotaxis across monolayers of GAG-deficient mutant cells. In summary, this study shows that multimerization is neither necessary for, nor potentiates, the biological activity of MCP-1. However, the results do clearly demonstrate the importance of the interaction between MCP-1 and cell-surface heparan sulphate for transmonolayer leucocyte chemotaxis.

The Ti-plasmid-encoded two-component sensor-regulator system comprising VirA and VirG confers upon Agrobucterium tumefuciens the ability to respond chemotactically to nanomolar concentrations of uir-inducing phenolics such as acetosyringone. Non-phosphorylatable, mutant VirA and VirG proteins are incapable of replacing their wild-type counterparts in conferring this phenotype. This indicates that, like vir-gene induction in response to acetosyringone, chemotaxis to the same ligand involves phosphorylation of VirA and VirG. However, unlike vir-induction, deletion of the periplasmic domain of VirA severely curtails acetosyringone chemotaxis, suggesting that acetosyringone may mediate effects through more than one region of VirA. When introduced into strains expressing wild-type VirA and VirG, the non-phosphorylatable versions suppress chemotaxis towards acetosyringone, implying that mutant copies of VirA and VirG compete with their wild-type counterparts in interactions between VirA and VirG.