A role for TRPV1 in agonist-evoked activation of human platelets.

BACKGROUND: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. OBJECTIVES: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. METHODS: Platelets loaded with fura-2 or sodium-binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5-HT secretion was determined by fluorescence assay after conjugation with o-phthaldialdehyde. ATP secretion was determined using luciferin-luciferase. RESULTS: TRPV1 was identified by Western blotting using a specific anti-hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca(2+) influx and Ca(2+) release from intracellular stores, responses that were blocked in a dose-dependent manner by the TRPV1 antagonists, 5'-Iodo-resiniferatoxin (5'-Iodo-RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na(+)]. Capsaicin-evoked Ca(2+) release was abolished in the absence of extracellular Na(+) or by the 5-HT(2A) receptor antagonist, ketanserin. Capsaicin evoked 5-HT release from platelets, a response abolished in the absence of extracellular Na(+) or by 5'-Iodo-RTX. Thus capsaicin-evoked Ca(2+) release appeared to be mediated by Na(+)-dependent 5-HT release. TRPV1-dependent 5-HT release also contributed to ADP- and thrombin-evoked Ca(2+) entry and release. 5'-Iodo-RTX reduced ADP- and thrombin-evoked Ca(2+) signals, effects not additive with those of ketanserin, and 5'-Iodo-RTX inhibited agonist-evoked 5-HT and ATP release. CONCLUSION: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis.

hTRPC1-associated alpha-actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation.

BACKGROUND: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store-operated Ca(2+) entry (SOCE). OBJECTIVES: To assess hTRPC phosphotyrosine content upon platelet stimulation. METHODS: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. RESULTS: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of alpha-actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr-evoked alpha-actinin tyrosine phosphorylation was increased by inhibiting the alpha-actinin phosphatase, SHP-1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca(2+) sensor STIM1. CONCLUSIONS: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.

Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita.

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis.

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Bovine beta-lactoglobulin at 1.8 A resolution--still an enigmatic lipocalin.

BACKGROUND: beta-Lactoglobulin (beta-Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine beta-Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants. RESULTS: We have redetermined the structure of beta-Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways. CONCLUSIONS: Incorrect threading of the sequence in the published structures of beta-Lg affects four of the nine beta strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the beta-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5 degrees, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the beta strand with the free thiol, each of which shows genetic variation.

Thermal denaturation of beta-lactoglobulin: effect of protein concentration at pH 6.75 and 8.05.

Previous work on the thermal denaturation of beta-lactoglobulin at about neutral pH and concentrations generally above 50 mg/ml has shown that the temperature of the maximum in the thermogram increases only slightly with concentration. Likewise, there is little if any concentration dependence at acid pH over a wide concentration range. However, so far as we are aware, no work has been described on the thermal denaturation of beta-lactoglobulin in the physiological range of protein concentration and pH appropriate to milk. We report measurements at pH 6.75 and 8.05 in the concentration range 2-120 mg/ml and show that below about 50 mg/ml the position of the maximum becomes strongly dependent on concentration, passing through a minimum near 25 mg/ml and increasing towards the lowest concentrations where measurements were practicable. Moreover, the narrow, well defined and nearly symmetrical thermal transition observed at high protein concentrations contrasts with a broader and more asymmetric curve at lower concentrations. An explanation for the behaviour seen at the lower protein concentrations is suggested, based on the temperature- and concentration-dependent dissociation of the beta-lactoglobulin dimer and an associated conformational transition. The position of the maximum in the thermogram has a marked dependence on the rate of heating down to the lowest rate investigated of 10 degrees C per hour, showing the importance of slow kinetic effects in the denaturation of this protein.