Andreas Schmarda

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Insight into the Structure-Function Relation of Chloride Channels

Chloride channels are highly selective transport proteins ubiquitously expressed in eukaryotic cells. Biophysical methods allow discrimination between several different types of chloride channels with

ICln: a chloride channel paramount for cell volume regulation.

Cell volume regulation is a ubiquitous cell regulatory mechanism based on meticulously controlled ion transport mechanisms. Keeping the absolute volume constant seems to be of the highest priority for most cells and is achieved at the expense of altered intracellular ion concentrations. We have been able to demonstrate that ICln, a chloride channel cloned from epithelial cells, is paramount for the ability of swollen cells to regulate their volume back to that under resting conditions. A unique feature of ICln is the distinct sensitivity of these channels for nucleotides and nucleoside analogues added to the extracellular fluid. In addition, cromolyn sodium and nedocromil sodium, drugs used by patients with asthma, are able to impede the function of these channels.

Blockade of swelling-induced chloride channels by phenol derivatives

1 . In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and NPPB (5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2 . RVD in NIH3T3 fibroblasts and MDCK cells is temperature‐dependent. 3 . RVD, the swelling‐dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4 . The cyclic AMP‐dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium‐activated chloride current in fibroblasts is insensitive. 5 . The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G × G × G, a glycine repeat located at the predicted outer mouth of the Icln channel protein.

The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels.

It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances – which are known to block the H,K‐ATPase – could also lead to an inhibition of swelling‐dependent chloride channels. Swelling‐dependent chloride channels – characterized in many different cell types – show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling‐dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling‐dependent chloride channels (IClswell). Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide‐bridges. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.

Determination of protein-protein interactions of ICIn by the yeast two-hybrid system.

ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B’.

Characterization of the human gene coding for the swelling-dependent chloride channel ICln at position 11q13.5–14.1 (CLNS1A) and further characterization of the chromosome 6 (CLNS1B) localization

Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.

Cloning of the Swelling-Dependent Chloride Channel lCln Homologue from Zebra Fish

Using expression cloning techniques, a protein was isolated which gave rise to an outwardly rectifying chloride current after expression in Xenopus laevis oocytes [Paulmichl et al., Nature 1992;356:238-241]. We termed this protein ICln and found it to be crucial for the regulatory volume decrease after swelling cells in hypotonic medium. Here we describe the cloning of the zebra fish homologue to ICln and, in addition, specify the epitope recognized by the antibodies we raised in rabbits against a synthetic peptide comprised by the C-terminal 24 amino acids of ICln·

The Promoter for Constitutive Expression of the Human ICln Gene CLNS1A

The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.