[
International C. elegans Meeting,
2001]
In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. This plays several documented roles: regulation of cytoplasmic chloride concentration, reduction of cell size in development, defense against cell swelling under hypoosmotic and isoosmotic conditions. The last, which includes the defense against swelling during moderate warming due to hyperactivity of the Na-K pump, has received the least attention but may be the most physiologically relevant. The gene sequences for two mammalian isoforms of this cotransporter were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (KO2A2.3, "KO2", on chromosome 2, and R13A1.2, "R13", on chromosome 4). Both C. elegans isoforms are expressed,as demonstrated by their presence in EST's and cDNA libraries. K02 has been shown to have K-Cl cotransport activity when transfected into mammalian cells. We have carried out dsRNAi blockages against both of these genes, singly and in combination, and found that about a third of the injected parents produced progeny that exhibit reduced survival at elevated temperature (33degC and 37degC) and increased sensitivity (seen as immobilization) to isoosmotic challenge induced by exposure to 100 or 200 mM ammonium chloride in M9 medium. A deletion mutant of K02 obtained from Gary Moulder at U. Oklahoma has not shown a phenotype by these tests and is now being used as a target for RNAi knockout of the R13 gene. GFP's expressed from upstream sequences of the two genes indicate that both isoforms are consistently expressed in pharyngeal muscle. The extent to which either is expressed elsewhere is still being determined. These studies may lead to establishing the role of K-Cl cotransporters in the whole organism and lay the foundation for a genetic analysis of their control