Inhibition of volume-regulated anion channels by dominant-negative caveolin-1.

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity.

Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains.

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I(Cl,swell), the swelling-induced Cl- current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I(Cl,swell). However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I(Cl,swell) in CPAE cells. Kinase activity was dispensable for the inhibition of I(Cl,swell), since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.

Cellular function and control of volume-regulated anion channels.

Restoration of cell volume after cell swelling in mammalian cells is achieved by the loss of solutes (K+, Cl-, and organic osmolytes) and the subsequent osmotically driven efflux of water. This process is generally known as regulatory volume decrease (RVD). One pathway for the swelling induced loss of Cl- (and also organic osmolytes) during RVD is the volume-regulated anion channel (VRAC). In this review, we discuss the physiological role and cellular control of VRAC. We will first highlight evidence that VRAC is more than a volume regulator and that it participates in other fundamental cellular processes such as cell proliferation and apoptosis. The second part concentrates on the Rho/Rho kinase/myosin phosphorylation cascade and on compartmentalization in caveolae as modulators of the signal transduction cascade that controls VRAC gating in vascular endothelial cells.

Enalapril in paediatric patients with Alport syndrome: 2 years' experience.

UNLABELLED: Enalapril, a long-acting inhibitor of angiotensin-converting enzyme, was given for 2 years to seven children with Alport syndrome. Five patients had a classical X-linked form of the disease; two siblings had the autosomal recessive variant. Their age was between 5.15 and 13.75 years when enalapril was started. All patients had haematuria and proteinuria, creatinine clearance was > 80 ml/min per 1.73 m2 in all, and only one patient was hypertensive. The starting dose of enalapril (0.1 mg/kg body weight per day) was increased progressively according to individual clinical tolerance. The median doses were 0.13, 0.12, 0.21 and 0.29 mg/kg at 6, 12, 18 and 24 months, respectively. Median values of mean blood pressure were 95 mmHg at the start and 84 mmHg after 24 months. Median daily proteinuria decreased from 52 mg/kg to 18 mg/kg at 6 months, 21 mg/kg at 12 months, 12 mg/kg at 18 months and 30 mg/kg at 24 months. Serum creatinine increased over time from a median of 0.64 mg/dl at baseline to 0.77 mg/dl at 24 months. Concomitantly, there was a decrease in GFR from 104 to 83 ml/min per 1.73 m2 at 18 months and an increase again to 95 ml/min per 1.73 m2 at 24 months. Analysis of the individual data showed three patterns: no response (n = 2), temporary response (n = 2) and sustained response (n = 3). CONCLUSION: When given enalapril at the dosages mentioned, Alport patients as a group display a marked reduction in urinary protein excretion with a nadir of 23% of the baseline figure at 18 months, a decrease that cannot be accounted for by the slight decrease in glomerular filtration rate. Although these are preliminary data, it is recommended to try an angiotensin-converting enzyme inhibitor in every paediatric Alport patient with proteinuria.

Caveolin-1 modulates the activity of the volume-regulated chloride channel.

1. Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). 2. ICl,swell, the cell swelling-induced chloride current through VRACs, was studied in three caveolin-1-deficient cell lines: Caco-2, MCF-7 and T47D. 3. Electrophysiological measurements showed that ICl, swell was very small in these cells and that transient expression of caveolin-1 restored ICl,swell. The caveolin-1 effect was isoform specific: caveolin-1beta but not caveolin-1alpha upregulated VRACs. This correlated with a different subcellular distribution of caveolin-1alpha (perinuclear location) from caveolin-1beta (perinuclear and peripheral). 4. To explain the modulation of ICl, swell by caveolin-1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs.

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells.

Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.

Functional effects of expression of hslo Ca2+ activated K+ channels in cultured macrovascular endothelial cells.

The aim of the present study is to elucidate the effects of the expression of large conductance Ca2+ activated K+ channels (BK[Ca]) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK(Ca) was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK(Ca). Membrane potential, ionic currents and Ca2+ signals were investigated in non-transfected and transfected cells using a combined patch clamp and Fura-2 fluorescence technique. In non-transfected control CPAE cells, ATP evoked a Ca2+ activated Cl- current (I[Cl,Ca]). The most prominent current component during ATP stimulation in hslo expressing cells was conducted BK(Ca) which resulted in a pronounced transient hyperpolarization. This hyperpolarization, which was absent in non-transfected cells, was enhanced if I(Cl,Ca) was blocked with niflumic acid. The sustained component of the Ca2+ response during ATP stimulation was significantly larger in hslo transfected cells than in non-transfected cells. This plateau level correlated well with the corresponding effects of ATP on the membrane potential, indicating that the expression of cloned BK(Ca) exerts a positive feedback on Ca2+ signals in endothelial cells by counteracting the negative (depolarizing)effect of stimulation of Ca2+-activated Cl- channels.