Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells.

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca(2+) transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5-20 µM) -induced Ca(2+) transients were blocked by capsazepine (CPZ) (10 µM), the TRPV4 activator 4α-PDD (10 µM) -induced Ca(2+) increases were reduced by ruthenium-red (RuR) (20 µM). Different heating (<40°C or >43°C) led to Ca(2+) increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca(2+) transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.

Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells.

The transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-and Mg(2+) permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca(2+) influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca(2+)](i) transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 µmol/l) as well as moderate heat (<40 °C) elicited [Ca(2+)](i) transients. Hypotonicity increased [Ca(2+)](i) and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca(2+) transients and underlying currents induced by known activators of this channel.

Thermosensitive transient receptor potential channels in human corneal epithelial cells.

Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca(2+). TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca(2+)](i) transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1-3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca(2+) transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10-20 µM), a non-selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40 °C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La(3+)) (100 µM) suppressed these temperature-induced Ca(2+) increases. Planar patch-clamp technique was used to characterize the currents underlying Ca(2+) transients. Increasing the temperature to over 40 °C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25%) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermosensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress.

Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.

COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.