ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the "ball and chain" model proposed for the Shaker potassium channel, i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.ICln is a protein that was identified by screening a cDNA library from Madin-Darby canine kidney (MDCK) 1 cells in Xenopus laevis oocytes using the two-electrode voltage-clamp technique (1). The expression of ICln in X. laevis oocytes results in an outwardly rectifying ion current that can be blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and the addition of nucleotides to the extracellular fluid. The kinetic, selectivity, and pharmacology of the ICln-induced currents resemble those of the anionic currents activated after cell swelling in a variety of cells (2). The activation of these channels permits the exit of ions, which in turn leads to the exit of water, therefore allowing an effective regulatory volume decrease (3). The molecular entity of the regulatory volume decrease-induced "anionic" channels (RVDCs) is still elusive. Our hypothesis that ICln is a candidate for RVDCs is supported by the fact that the selective knockdown of the ICln protein in fibroblasts and epithelial cells leads to a substantial decrease in swellinginduced RVDC activation (4, 5). Furthermore, t...

No abstract

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.