The Molecular Function Of Mclca1, Mclca2 And Mclca4 In Murine Life

Abstract

Chloride channel, calcium-activated proteins (CLCAs) have been reported to regulate chloride transport and be involved in the pathophysiology of diseases, such as cystic fibrosis, asthma, airway inflammation and cancer. They have been cloned or isolated from multiple species, incluing human, mouse, bovine, equine, rat, porcine and canine. The aim of this research was to understand the function of this group of genes in murine life, focusing on mCLCA4 and its highly homologous family members, mCLCA1 and mCLCA2. mCLCA4 is one of six members in the murine genome cloned to date, and is highly expressed in smooth muscle. To begin to understand the function of this gene family, I investigated the cellular processing and regulatory sequences of mCLCA4 proteins. The full length mCLCA4 gene product [125 kilo-Dalton (kD)] is made in the endoplasmic reticulum and is cleaved to 90 kD and 40 kD fragments. Both fragments, 90 kD N- terminal and 40 kD C- terminal fragments are secreted out of the cell and associate with the cell membrane. A specific diarginine motif is the retention signal while a dileucine motif is the forward trafficking signal during mCLCA4 secretion. While secretion of mClCA4 excludes this gene product as a channel protein, its association with the membrane may be consistent with a role as a regulator of chloride conductance. To further understand mCLCA4 gene function, I generated mCLCA4 knock- out mice. These mice displayed no gross phenotype and bred normally. Specific lung challenge experiments are being undertaken by collaborators to examine the effect of airway challenge on mClCA4 null mice. To further study the function of mCLCA4 in vivo, mCLCA1, mCLCA2, mCLCA4 triple knock-out mice were made. A 112 kilo base pair (kb) sequence was deleted from chromosome 3 of the murine genome using bacterial artificial chromosomal (BAC) recombineering techniques. Quantitative PCR was used to screen for positive embryonic stem cell clones that were then injected into blastocysts using standard techniques. Highly chimeric mice were bred to C57Bl/6J mice to produce heterozygous offspring. Currently, the triple gene knock-out mice survive to birth, but further phenotypic evaluation is needed.