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B Nilius

Publications and source records attributed to B Nilius.

At least 37 records · Page 2Linked to original sources
PubMed2001

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G).

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.

Aspartic Acid↗
PubMed2001

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.

Animals↗
PubMed2001

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Animals↗
PubMed2001

Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells.

We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.

Animals↗
PubMed2001

The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel.

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.

Amino Acid Sequence↗
PubMed2001

The amino side of the C-terminus determines fast inactivation of the T-type calcium channel alpha1G.

We analysed the kinetic properties of the fast inactivating T-type calcium channel alpha1G in HEK 293 cells transfected with different alpha1G chimeras, containing the N-terminus, III-IV linker or various C-terminal regions of the slowly inactivating L-type alpha1C. A highly negatively charged region of 23 amino acids at the amino side of the intracellular carboxy terminus of alpha1G was found to be critical for fast inactivation. The N-terminus of alpha1G does not seem to be necessary for inactivation of the T-type calcium channel because replacement of the a1G N-terminus with the alpha1C N-terminus did not influence channel kinetics at all. Replacing the III-IV linker of alpha1G with that of a1C decreased the rate of inactivation at -20 mV from 15.8 +/- 1.8 to 8.5 +/- 1.1 ms, and shifted the potential for half-maximal inactivation from -69.6 +/- 0.8 to -54.0 +/- 1.7 mV. However, these parameters were not significantly different at other potentials. We suggest a putative 'ball-and-chain'-like mechanism for inactivation in which the negative charges function as an acceptor domain for a ball, hypothetically located at a different intracellular part of the channel. In addition, transferring the IQ motif and EF hand of alpha1C to alpha1G does not confer Ca2+-dependent inactivation on alpha1G, suggesting that other sequences besides the C-terminus are needed for Ca2+-dependent inactivation of alpha1C.

Algorithms↗
PubMed2001

Modulation of the epithelial Ca2+ channel ECaC by extracellular pH.

We investigated the effect of extracellular pH on whole-cell currents through the epithelial Ca2+ channel, ECaC, expressed in HEK 293 cells. Both mono- and divalent current densities were significantly smaller at pH 6.0 than at pH 7.4. At pH 8.5 they were slightly larger. Lowering extracellular pH enhanced the slow component of monovalent current activation at negative potentials but had no significant effect on the kinetics of Ca2+ currents. The kinetics of block of monovalent cation current by extracellular Mg2+ was significantly changed at high and low pH. The time constant of the time- and voltage-dependent current component during a voltage step to -140 mV was significantly larger at pH 8.5 than at pH 7.4. At pH 6.0 it was almost absent. The [Mg2+] inhibiting 50% of monovalent current through ECaC at pH 6.0 (IC50) was 323 +/- 23 microM (n = 8), compared with 62 +/- 9 microM (n = 4) at pH 7.4 and 38 +/- 4 microM (n = 8) at pH 8.5. The affinity of ECaC for Ca2+ was also affected by extracellular pH, shifting from 4.8 +/- 0.7 microM (n = 6) at pH 6.0 to 161 +/- 30 nM (n = 5) at pH 7.4 and 425 +/- 117 nM (n = 8) at pH 8.5.

Calcium↗
PubMed2001

Ca(2+)-activated Cl- channels in Ehrlich ascites tumor cells are distinct from mCLCA1, 2 and 3.

Using the whole-cell patch-clamp technique, we have studied the electrophysiological and pharmacological properties of the Ca(2+)-activated Cl- current present in Ehrlich cells. The currents activated slowly upon depolarization, deactivated upon hyperpolarization, and showed strong outward rectification. An increase in [Ca2+]i activated the current with an EC50 of 165.2 nM. Extracellular application of niflumic acid (100 microM) rapidly blocked the current in a voltage-dependent manner whereas sulfhydryl-modifying agents such as dithiothreitol (DTT, 1-2 mM) and N-ethylmaleimide (NEM, 100 microM) had no effect on Ca(2+)-activated currents in Ehrlich cells. Members of the recently discovered CLCA gene family are the only molecular candidates for Ca(2+)-activated Cl- channels cloned so far. Using RT-PCR we demonstrated that the appearance of a Ca(2+)-activated Cl- current in Ehrlich cells is not associated with the expression of the murine members of the CLCA family (mCLCA1-mCLCA3). Correspondingly, the kinetic and pharmacological properties of the Ca(2+)-activated current in Ehrlich cells differ from those of CLCA-associated currents, which are time independent and DTT sensitive. Thus, phenotypic differences in combination with RT-PCR data point to the existence of different molecular species for Ca(2+)-activated Cl- channels.

Animals↗
PubMed2001

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Amino Acid Motifs↗
PubMed2001

Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volume-regulated anion currents in HEK-293 cells.

The detection of changes in volume and osmolality is an essential function in vertebrate cells. A novel member of the transient receptor potential (trp) family of ion channels, which is sensitive to changes in cell volume, has been described recently. Heterologous expression of TRP12 in HEK cells resulted in the appearance of a swelling-activated cation current. The permeability sequence of this cation current for various monovalent cations, as determined from shifts in reversal potential upon extracellular cation substitution, was PK>PCs>PNa>PLi, corresponding to an Eisenman-IV sequence characteristic for a weak-field-strength site. Surprisingly, over-expression of this channel in HEK cells was accompanied by a dramatic down-regulation of the volume-regulated anion channel (VRAC), which is activated by cell swelling in non-transfected cells. In contrast to VRAC, TRP12 could not be activated at constant volume by a reduction of intracellular ionic strength or by intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate (GTPgammaS). The kinetic and pharmacological profile of VRAC and TRP12 currents were also different.

Anions↗
PubMed2001

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Acetylcholine↗
PubMed2001

Inhibition of volume-regulated anion channels in cultured endothelial cells by the anti-oestrogens clomiphene and nafoxidine.

1. We have used the whole-cell patch clamp technique to study the effect of the partial anti-oestrogens clomiphene and nafoxidine, the pure anti-oestrogens ICI 182,780 and RU 58,668 and the oestrogen ss-estradiol, on the volume-regulated anion channel (VRAC) in cultured pulmonary artery endothelial (CPAE) cells. 2. In contrast to the pure anti-oestrogens and ss-estradiol, clomiphene and nafoxidine potently inhibited the volume-sensitive chloride current, I(Cl,swell), activated by challenging CPAE cells with a 25% hypotonic solution. For clomiphene, the estimated IC(50) and Hill coefficient were 1.03+/-0.14 microM and 1.40+/-0.21 respectively. In the case of nafoxidine, these values were 1.61+/-0.29 microM and 1.24+/-0.19. 3. The inhibition induced by the pure enantiomers of clomiphene, zuclomiphene and enclomiphene, was not different from that of the racemic mixture, indicating that the interaction between clomiphene and VRAC is not stereoselective. 4. Clomiphene and nafoxidine inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.98+/-0.17 and 1.66+/-0.21 microM respectively, concentrations similar to those for half-maximal block of VRAC. 5. In conclusion, the nonsteroidal partial anti-oestrogens nafoxidine and clomiphene are potent inhibitors of volume-regulated anion channels. The inhibition by clomiphene is not stereoselective and occurs at concentrations close to therapeutically relevant concentrations. Finally, both drugs inhibit the proliferation of endothelial cells.

Cell Division↗
PubMed2001

Pharmacological modulation of monovalent cation currents through the epithelial Ca2+ channel ECaC1.

1. The recent identification of the epithelial Ca(2+) channel, ECaC1, represents a major step forward in our knowledge of renal Ca(2+) handling. ECaC1 constitutes the rate-limiting apical Ca(2+) entry mechanism of active, transcellular Ca(2+) reabsorption. This unique highly selective Ca(2+) channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR). 2. We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca(2+) dependent inactivation of ECaC1. 3. Several modulators were tested, including inorganic cations, putative store-operated Ca(2+) entry (SOC) blockers, the vanilloid receptor (VR-1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red. 4. Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC(50) values of 111 nM and 1.3 microM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective. 5. The divalent cation current block profile for ECaC1 is Pb(2+)=Cu(2+) >Zn(2+) >Co(2+) >Fe(2+) with IC(50) values between 1 and approximately 10 microM. 6. In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca(2+) homeostasis could benefit from such developments.

Antifungal Agents↗
PubMed2001

DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration.

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.

Action Potentials↗
PubMed2001

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+.

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.

Calcium↗
PubMed2001

Endothelial intracellular Ca2+ release following monocyte adhesion is required for the transendothelial migration of monocytes.

Although molecular changes accompanying leukocyte extravasation have been investigated intensively, the particular events following leukocyte adhesion and leading to the actual transendothelial migration process remain largely unknown. To characterize intraendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca(2+)levels ([Ca(2+)]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca(2+)transients in either untreated or TNF-alpha-stimulated microvascular endothelial cells (HMEC-1). The functional significance of these [Ca(2+)]i rises was demonstrated by treating filter-grown endothelial monolayers with BAPTA/AM. This in traendothelial Ca(2+)chelation left monocyte adhesion basically unaffected, but caused a significant and dose-dependent reduction of the transendothelial migration of monocytes. Granulocyte diapedesis, on the other hand, was hardly modified. Thapsigargin-treatment of endothelial cells almost completely inhibited the transmigration of monocytes suggesting that the necessary Ca(2+)transients depended on a release from intracellular Ca(2+)stores. Our results thus show that the transmigration of monocytes through endothelial monolayers of microvascular origin is favoured by an increase of the intraendothelial [Ca(2+)]i induced by leukocyte adhesion to the endothelial cells.

Calcium↗
PubMed2001

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.

Acylation↗