Our goals are to characterize basolateral Cl-channels from rabbit and mouse medullary thick limbs (MTAL) and from mouse cortical thick limbs (CTAL). ADH enhances net NaCl absorption in microperfused MTAL but not CTAL. In the MTAL, ADH may increase apical salt admittance, and the rise in cell Cl- may increase basolateral Cl- conductance secondary. We previously studied Cl-channels from basolateral vesicles of rabbit outer medulla and expressed a Cl- conductance in Xenopus oocytes using mRNA from rabbit outer medulla. Current progress included: A) Characterizing Cl-channels from rabbit outer medulla and from cultured mouse MTAL and CTAL cells; B) Identifying a putative Cl-channel cDNA, rbCIC-Ka, from rabbit outer medulla; C) Providing evidence that rbCIC-Ka, the 75 kDa gene product of rbCIC-Ka, mediates salt absorption in the MTAL; D) Comparing the properties of Cl-channels in cultured mouse MTAL and cultured mouse CTAL cells; E) Studying the kinetics of Cl-flux through MTAL Cl- channels.
Our aims are: (i) To express rbCIC-Ka. (ii) To understand the molecular physiology of basolateral Cl- channels in mouse MTAL and CTAL. (iii) To show that the Cl-conductance increase produced when Xenopus oocytes are infected with 1.8-3.2 kb mRNA from rabbit outer medulla is due to rbCIC-Ka. (iv) We have isolated two highly homologous cDNAs, mmCIC-Ka and mmCIC-Ka, from mouse medulla and cortex, respectively. We wish to establish whether mmCIC-Ka and mmCIC-Ka encode Cl-channels mediating net Cl-absorption in mouse MTAL and CTAL, respectively; and to express the gene products of mmCIC-Ka and mmCIC-Ka. (v) To evaluate which proposed phosphorylation sites on the cytosolic surfaces of rbCIC-Ka, mmCIC-Ka and mmCIC-Ka (the proposed proteins encoded by rbCIC-Ka, mmCIC-Ka and mmCIC-Ka, respectively) are phosphorylated by (ATP + PKA). (vi) To evaluate why anti-rbCIC-Ka, prepared using the cytoplasmic C-terminus of rbCIC-Ka as antigen, blocks rbCIC-Ka channels at extracellular faces. (viii) To evaluate the kinetics of Cl-permeation in Cl-channels from mouse CTAL. These data may help provide a molecular basis for the functional differences between basolateral MTAL and CTAL Cl-channels. The results may help explain the molecular basis for some salt-acquisitive or salt-wasting disorders., and allow the rational design of drugs which ameliorate such derangements.
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