Search PubMed⌕ Search

Biomedical subjects

B Nilius

Publications and source records attributed to B Nilius.

At least 91 records · Page 5Linked to original sources
PubMed1998

Characterization of mutations located in exon 18 of the CFTR gene.

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

Animals↗
PubMed1998

The annexin II-p11 complex is involved in regulated exocytosis in bovine pulmonary artery endothelial cells.

Annexin II is a member of a multigene family of Ca2+-regulated, membrane-binding proteins implicated through biochemical and perforated cell experiments in Ca2+-triggered secretion. Within most cells annexin II resides in a tight heterotetrameric complex with a cellular protein ligand, p11, and complex formation is mediated via the N-terminal 14 residues of annexin II including the N-terminal acetyl group. To analyze at the single cell level whether the annexin II-p11 complex is involved in regulated secretion, we used membrane capacitance measurements to follow exocytotic fusion events in bovine aortic endothelial cells manipulated with respect to their annexin II-p11 complex formation. Upon guanosine 5'-O-(thiotriphosphate) (GTPgammaS) stimulation, the endothelial cells show a significant increase in membrane capacitance which is generally preceded by a transient rise in intracellular Ca2+ and thus indicative of the occurrence of Ca2+-regulated secretion. The GTPgammaS-induced capacitance increase is markedly reduced in cells loaded with a synthetic peptide, Ac1-14, which corresponds in sequence to the N-terminal 14 residues of annexin II in their correctly acetylated form and which is capable of disrupting preformed annexin II-p11 complexes. The effect of the peptide is highly specific as the nonacetylated variant, N1-14, which is incapable of disrupting annexin II-p11, does not interfere with the GTPgammaS-induced increase in membrane capacitance. These data show that intact annexin II-p11 complexes are indispensable for regulated exocytosis to occur in an efficient manner in endothelial cells.

Animals↗
PubMed1998

Separate swelling- and Ca2+-activated anion currents in Ehrlich ascites tumor cells.

A Ca2+-activated (ICl,Ca) and a swelling-activated anion current (ICl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca2+]i), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl- concentration. ICl,Ca current density increased with increasing [Ca2+]i, and this current was abolished by lowering [Ca2+]i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). In contrast, activation of ICl,vol did not require an increase in [Ca2+]i. The kinetics of ICl,Ca and ICl,vol were different: at depolarized potentials, ICl,Ca as activated in a [Ca2+]i- and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, ICl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of ICl, vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I- > Cl- > gluconate. ICl,Ca was inhibited by niflumic acid (100 micron), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 micron) and 4, 4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 100 micron), niflumic acid being the most potent inhibitor. In contrast, ICl,vol was unaffected by niflumic acid (100 micron), but abolished by tamoxifen (10 micron). Thus, in Ehrlich cells, separate chloride currents, ICl,Ca and ICl,vol, are activated by an increase in [Ca2+]i and by cell swelling, respectively.

Animals↗
PubMed1998

The GXGXG motif in the pI(Cln) protein is not important for the nucleotide sensitivity of the pI(Cln)-induced Cl- current in Xenopus oocytes.

It has been proposed that the pI(Cln) protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pI(Cln) in which the three glycines of the putative nucleotide-binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage-dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage-dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide-binding site in pI(Cln).

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins.

1. The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl- current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5% (mild hypotonicity) or 25% (strong hypotonicity). 2. The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 +/- 0.2, 61.4 +/- 1.7 and 62.9 +/- 1.3 microM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 microM) and dephostatin (20 microM) potentiated ICl,swell activated by mild hypotonicity by 47 +/- 9 and 69 +/- 15%, respectively. 4. Intracellular perfusion with GTP gamma S (100 microM) transiently activated a Cl- current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity-induced cell shrinkage completely inhibited the GTP gamma S-activated Cl- current. 5. Intracellular perfusion with GDP beta S (1 mM) caused a time-dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6. Our results demonstrate that the activity of endothelial swelling-activated Cl- channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Action Potentials↗
PubMed1998

Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells.

1. We have studied the effects of intracellular ionic strength (gamma 1) on the swelling-activated whole-cell Cl- current (ICl,swell) in cultured calf pulmonary artery endothelial cells (CPAE cells). 2. Reducing gamma 1 from 155 to 95 mM at constant osmolarity and Cl- concentration activates an outwardly rectifying current that is mainly carried by Cl- ions and inactivates at positive potentials. The amplitude of the current is larger at more reduced levels of gamma 1. 3. The permeability ratio for the anions I-, Br-, Cl- and gluconate (PI: PBr: PCl: Pgluc) was 1.35:1.03:1:0.17. 4. Blockers of the swelling-activated Cl- current in CPAE cells also inhibit the current which is activated by a reduction in gamma 1 with an IC50 of 1.1 microM for tamoxifen, 1.3 microM for mibefradil, and 35 microM for quinidine. 5. The protein tyrosine kinase inhibitors tyrphostin B46 (50 microM) and genistein (100 microM), which inhibit ICl,swell in CPAE cells, also inhibited the gamma 1-induced current by 92.9 +/- 2.4% (n = 3) and 41.2 +/- 5.0% (n = 4), respectively. 6. Hypertonic extracellular solutions rapidly and reversibly antagonized the gamma 1-activated current, whereas increasing gamma 1 from 155 to 195 mM precluded activation of ICl,swell by hypotonic shock. 7. It is concluded that a reduction of gamma 1 activates an anion current that is identical to that activated by cell swelling. Changes in intracellular ionic strength may shift the volume set point for activation of ICl,swell.

Action Potentials↗
PubMed1998

Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.

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.

Alleles↗
PubMed1998

Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl- currents in macrovascular endothelial cells.

We have used the whole-cell patch-clamp technique to study the effects of inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] and inositol 1,3, 4,5,6-pentacisphosphate [Ins(1,3,4,5,6)P5] on volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary artery (CPAE cells). Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 were applied intracellularly via the patch pipette at concentrations between 10 and 100 muM. Both tetrakisphosphates inhibited the Cl- current ICl,Ca, which was activated by intracellular loading of the cells with 500 nM Ca2+ [for inhibition by Ins(1,4,5,6)P4: 58% at 10 muM, 75% at 100 muM; for Ins(3,4,5,6)P4: 44% at 10 muM, 65% at 100 muM]. Inhibition of ICl,Ca occurred without significant changes in its kinetic properties. The amplitude of ICl,vol activated by a 13.5 or 27% hypotonic solution at +100 mV was strongly reduced in cells loaded with either tetrakisphosphate, i.e. a 73% reduction for Ins(3,4,5,6)P4 and 89% for Ins(1,4,5,6)P4 at 100 muM. Both tetrakisphosphates also inhibited a current probably identical to ICl,vol which was activated by dialysing the cell with 100 muM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]). Ins(1, 3,4,5,6)P5 at a concentration of 30 muM did not significantly reduce ICl, vol. The effects of Ins(3,4,5,6)P4 may represent an inhibitory pathway for the ICl,Ca and ICl,vol in macrovascular endothelium after sustained receptor-mediated activation of phospholipase C.

Animals↗
PubMed1998

Store depletion triggers the calcium release-activated calcium current (ICRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines.

In endothelial cells, different types of Ca2+ conductances have been described, but none of them has been clearly identified as ICRAC, the Ca2+ release-activated Ca2+ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate (InsP3), Ca2+ ionophores and Ca2+ pump inhibitors activates a Ca2+-selective conductance in the presence of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La3+. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an ICRAC-like current. At 0 mV holding potential the estimated current density is -0.1 and -0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na+ currents are detectable in CPAE and HEK cells following store depletion in Ca2+- and Mg2+-free medium. The current carried by Na+ ions is similarly blocked by micromolar La3+, is inwardly rectifying and has a positive reversal potential.

Animals↗
PubMed1998

Modulation of volume-regulated anion channels by extra- and intracellular pH.

We have studied the modulation of the volume-regulated anion channel (VRAC) in cultured endothelial cells from bovine pulmonary artery (CPAE cells) by extra- and intracellular pH. The patch-clamp technique was used in combination with a fluorimetric measurement of intracellular pH using BCECF. Swelling of CPAE cells was accompanied by a slow acidification. The metabolites lactate and HCO3- both permeate through VRAC. The inactivation of VRAC currents at positive potentials is accelerated at a decreased extracellular pH and decelerated at alkaline pH. The instantaneous current amplitude is only slightly affected. Intracellular alkalization reduced whereas acidification enhanced the currents flowing through VRAC at all potentials. HCO3- and lactate permeation, as well as the up-regulation of VRAC at an acidic intracellular pH might be related to a possible role of this channel in cellular pH regulation.

Animals↗
PubMed1998

Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular endothelium.

Microfluorometric measurements in Fura-2-loaded single cultured human vascular endothelial cells were used to characterize the intracellular calcium [Ca2+]i responses triggered by extracellular application of adenosine 5'-triphosphate (ATP) and other nucleotides. Application of ATP or uridine 5'-triphosphate (UTP) gave rise to dose-dependent elevations of [Ca2+]i in all the cells tested. At saturating concentrations of agonist, the [Ca2+]i response was biphasic, with an early peak and a sustained plateau. Unlike peak responses, the sustained Ca2+ plateau was sensitive to removal of Ca2+ from the external medium. Mn2+ quenching revealed the presence of Ca2+ influx during the agonist-induced calcium plateau. The agonist-evoked calcium plateau was inhibited in a dose-dependent manner by the Cl-channel blocker NPPB, by the divalent cation Ni2+ and by the imidazole antimycotic econazole. Previously, these compounds have been shown to block store-operated Ca2+ entry. The two phases of the agonist-evoked [Ca2+]i response were blocked by the specific phospholipase C inhibitor U-73122 and by intracellular injection of low molecular weight heparin, suggesting the involvement of IP3-sensitive intracellular Ca2+ stores. The pharmacological profile of the response, using different nucleotides and analogues, ATP = UTP > ADP = UDP, and no responses to P2X1 and P2Y1 agonists, suggested the involvement of P2Y2 receptors. The expression of mRNA for the P2Y2 receptor was detected by RT-PCR analysis. These results indicate that P2Y2 receptors linked to intracellular Ca2+ mobilization are present in human vascular endothelial cells. The initial [Ca2+]i mobilization is followed by a phase of elevated [Ca2+]i influx.

Adenosine Triphosphate↗
PubMed1998

Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator.

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

Amino Acid Sequence↗
PubMed1998

Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes.

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 +/- 2.1 pS (n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 microM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

Animals↗
PubMed1998

Signaltransduction in vascular endothelium: the role of intracellular calcium and ion channels.

Endothelial cells (ECs) provide an ideal surface for blood flow. They inhibit the initiation of blood-clotting, but can also under certain conditions activate this process. ECs influence thrombolysis as well as thrombogenesis. They are "antigen-presenting cells" and play a key role in angiogenesis. In addition, ECs control the permeability of the barrier between bloodvessels and interstitium. One of their most important functions is the regulation of the diameter of the blood vessels and their adaptation to the demanded hemodynamic needs. The production and release of diverse compounds, which interfere with different neighboured target cells, initiate this plethora of functions. Ca2+ signals in endothelial cells play the key role in the release of NO, prostacyclin (PGI2), platelet activating factor (PAF), von Willebrand factor (vWF), tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI). Changes in the intracellular Ca2+ concentration ([Ca2+]i) are determined by release from intracellular stores and entry through the plasma membrane. The diversity of Ca2+ entry pathways and mechanisms of their control are described. At least two different types of Ca2+ entry channels exist: 1. typical highly Ca2+ selective ion channels which might be activated by depletion of intracellular Ca2+ stores (Ca2+ release-activated Ca2+ channels, CRAC), and 2. Non-selective Ca2+ permeable cation channels (NSC). The latter shares many features with an NSC induced by expression of the protein TRPC3. These channels are only weakly operated by store depletion and require a permissive Ca2+ and Ins(1,4,5)P3 concentration in the cytosol. CRAC channels are possible indirectly involved in Ca2+ entry during mechano-stimulation of ECs. After activation of these entry channels, influx of Ca2+ depends on the driving force. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identified as Kir2.1, a large-conductance, Ca2+ activated K+ channel (hslo) and at least two Cl- channels (a volume regulated Cl- channel, VRAC, and a Ca2+ activated Cl- channel, CaCC). It will be explained how these ion channels interact in the regulation of the long-lasting (plateau-type) increase in [Ca2+]i which mainly controls NO-synthesis and release. Furthermore, it will be demonstrated that Ca2+ oscillations depend on intracellular events rather than Ca2+ entry from the extracellular space.

Animals↗
PubMed1997

Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells.

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.

Adenosine Triphosphate↗
PubMed1997

Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific.

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids) corresponds to the previously published ClC-6 protein [Brandt and Jentsch (1995) FEBS Lett. 377, 15-20] and it has a canonical ClC structure. However, ClC-6b (320 amino acids), ClC-6c (353 amino acids) and ClC-6d (308 amino acids) are truncated at their C-termini. Hydropathy-plot analysis indicates that the shortened isoforms contain maximally four (ClC-6b and -6d) or seven (ClC-6c) transmembrane domains. Sequence analysis of a human genomic ClC-6 fragment indicates that the cDNA variability arises from alternative splicing at two different positions: the first alternative site consists of an intron flanked by two alternative donor sites and two alternative acceptor sites, the second being due to an exon that is optionally included or excluded. Reverse-transcription-PCR analysis of ClC-6 expression in human cell lines and tissues shows that the majority (83%) of ClC-6 mRNAs consists of ClC-6a or ClC-6c messengers. Furthermore, in a mouse tissue panel, the ClC-6a mRNA has a relatively broad tissue expression pattern, since it could be detected in brain, kidney, testis, skeletal muscle, thymus and pancreas. In contrast, expression of ClC-6c is more restricted, since it was only detected in kidney.

Alternative Splicing↗