The kidney is a major regulatory organ of calcium homeostasis. Perfused tubule studies have revealed that the component of calcium reabsorption which is active and hormonally-regulated resides largely in the distal convoluted tubule (DCT). Calcium is thought to enter cells of this segment through a specific apical channel. This process is the rate limiting step of calcium reabsorption, and is subject to hormonal regulation. The overall goal of this project is to provide a molecular description of the renal apical calcium channel involved in active calcium reabsorption. Specifically, the proposal is divided into the following aims: 1) Complete the cloning of a full-length cDNA encoding a voltage-dependent calcium channel alpha1-subunit previously identified as specific to the DCT; identify and clone the channel subunits associated with this molecule; and search for novel calcium channel-encoding transcripts expressed selectively in the DCT. 2) Determine the site(s) of expression within the kidney of all the cloned calcium channel subunits and isoforms identified in 1) above. 3) Study the properties of the proteins encoded by the cloned calcium channel isoforms described above. 4) Study the regulation of expression of the various cloned species under conditions of altered calcium homeostasis. The experimental techniques will include standard molecular cloning and sequencing strategies, using polymerase chain reaction (PCR) for cloning, microdissected tubule-PCR, Northern and Southern blotting, the generation antibodies against fusion proteins, immunohistochemistry, the functional expression of cDNA clones, and antisense oligonucleotide hybrid arrest strategy.
