Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca(2+) signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI(2). In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca(2+)-entry pathways or stabilize the driving force for Ca(2+) influx through these pathways. These Ca(2+)-entry pathways comprise agonist-activated nonselective Ca(2+)-permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca(2+) channels or capacitative Ca(2+) entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca(2+) entry is mainly controlled by large-conductance Ca(2+)-dependent BK(Ca) channels (slo), inwardly rectifying K(+) channels (Kir2.1), and at least two types of Cl( -) channels, i.e., the Ca(2+)-activated Cl(-) channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca(2+) signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.

TRPV4 is a Ca 2؉ -and Mg 2؉ -permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca 2؉ -dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5 ,6 -epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A 2 (PLA2) and cytochrome P450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4␣-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4␣-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA 2-dependent formation of AA, and its subsequent metabolization to 5 ,6 -epoxyeicosatrienoic acid by means of a cytochrome P450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA 2-and cytochrome P450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.T he TRPV subfamily of the transient receptor potential (TRP) family of cation channels consists of at least six mammalian channels homologous to the vanilloid receptor (for a unifying nomenclature, see ref. 1). The TRPV channels are activated by a variety of signals, including chemical and thermal stimuli, cell swelling, low intracellular Ca 2ϩ , and endogenous or synthetic ligands (2-10). Members of this subfamily contain a hydrophobic core region comprising six putative transmembrane segments (TM1-TM6), a pore-loop region between TM5 and TM6, a cytoplasmic N terminus with three to six ankyrin repeats, and a cytoplasmic C terminus (1, 3). The TRPV subfamily can be subdivided into two groups. One group is formed by TRPV1-TRPV4, which display a moderate Ca 2ϩ selectivity (P Ca ͞P Na Ͻ 10, in which P is permeability), a weak field-strength monovalent cation permeability sequence, and steep temperature dependence (5,6,(11)(12)(13)(14)(15)(16). The second group is formed by TRPV5 and TRPV6, which are highly Ca 2ϩ selective (P Ca ͞P Na Ͼ 100) and display a permeability sequence for monovalent cations consistent with a strong field-strength binding site but show little temperature dependence (10,17,18).TRPV4 was identified originally as a channel activated by hypotonic cell swelling (11,13,19), but later reports show that it can be activated also by synthetic agonists, such as the phorbol ester 4␣-phorbol 12,13-didecanoate (4␣-PDD) (5), temperatures Ͼ27°C (6, 20), and acidic pH and citrate (ref. 21; see also ref. 5). Moreover, rec...

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

This review describes molecular and functional properties of the following Cl- channels: the ClC family of voltage-dependent Cl- channels, the cAMP-activated transmembrane conductance regulator (CFTR), Ca2+ activated Cl- channels (CaCC) and volume-regulated anion channels (VRAC). If structural data are available, their relationship with the function of Cl- channels will be discussed. We also describe shortly some recently discovered channels, including high conductance Cl- channels and the family of bestrophins. We illustrate the growing physiological importance of these channels in the plasma membrane and in intracellular membranes, including their involvement in transepithelial transport, pH regulation of intracellular organelles, regulation of excitability and volume regulation. Finally, we discuss the role of Cl- channels in various diseases and describe the pathological phenotypes observed in knockout mice models.

1. The epithelial Ca(2+) channel (ECaC) family represents a unique group of Ca(2+)-selective channels that share limited homology to the ligand-gated capsaicin receptors, the osmolarity-sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated that this family is restricted to two members, ECaC1 and ECaC2 (also named CaT1). 2. RT-PCR analysis demonstrated that the two channels are co-expressed in calbindin-D-containing epithelia, including small intestine, pancreas and placenta, whereas kidney and brain only express ECaC1 and stomach solely ECaC2. 3. From an electrophysiological point of view, ECaC1 and ECaC2 are highly similar channels. Differences concern divalent cation permeability, the kinetics of Ca(2+)-dependent inactivation and recovery from inactivation. 4. Ruthenium red is a potent blocker of ECaC activity. Interestingly, ECaC2 has a 100-fold lower affinity for ruthenium red (IC(50) 9 +/- 1 microM) than ECaC1 (IC(50) 121 +/- 13 nM). 5. ECaCs are modulated by intracellular Mg(2+) and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg(2+) concentrations. 6. In conclusion, ECaC1 and ECaC2 are homologous channels, with an almost identical pore region. They can be discriminated by their sensitivity for ruthenium red and show differences in Ca(2+)-dependent regulation.

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Low concentrations of inositol 1,4,5-trisphosphate (InsP3) evoke a very rapid mobilization of intracellular Ca2+ stores in many cell types, which can be followed by a further, much slower efflux. Two explanations have been suggested for this biphasic release. The first proposes that the Ca2+ stores vary in their sensitivity to InsP3, and each store releases either its entire contents or nothing (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP3, but that they can release only a fraction of their Ca2+ before their sensitivity is somehow attenuated (steady-state release). Experiments using purified InsP3 receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP3 under conditions in which the total Ca2+ level at both sides of the receptor is held constant. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca(2+)-free medium, the amount of 45Ca2+ remaining in the stores after the rapid transient phase of release is independent of their initial Ca2+ levels, indicating that partially depleted stores are less sensitive to InsP3. Moreover, if the stores are reloaded with 40Ca2+ after the first stimulus, reapplication of the same low concentration of InsP3 will release further 45Ca2+. This recovery of InsP3 sensitivity is almost complete. Under these conditions, Ca2+ release must thus occur by a steady-state mechanism, in which the decreasing Ca2+ content of the stores slows down further release.

3. At the same [Ca]. the degree of filling is higher in K-depolarized tissues than in control tissues. However at 10 mM- [Ca]. and 5-9 mM-K the amount of Ca taken up by the store is larger than that after loading in 0-2 mM-Ca and 141-4 mM-K, although the tissues remain relaxed during loading at 5 9 mM-K and contracted at 141-4 mM-K.4. The Ca antagonists D600 and nicardipine selectively block the contraction induced by K depolarization, but do not affect appreciably the noradrenaline-induced contraction.5. The filling of the store is not significantly reduced by the presence of the Ca antagonists in solutions containing 5-9 mM-K. However these antagonists reduce the degree of filling in K-rich loading solution to a level which is lower than that observed in the control.6. Mn blocks both the contraction induced by K-rich solution and the tonic component of the noradrenaline-induced contraction and it also inhibits filling of the store.7. The results suggest that the filling of the store under physiological conditions occurs by a direct pathway between the store and the extracellular medium.