The bulk of filtered CI is reabsorbed in the proximal tubule by passive and active pathways. SLC26A6 has recently emerged as the likely key transporter that accounts for reabsorption of this active component, and thus contributes significantly to proximal tubule NaCI absorption and overall renal NaCI and extracellular volume homeostasis. The general project goal is to elucidate the molecular mechanisms regulatingSLC26A6. Preliminary studies by the applicant have indicated that activity of SLC26A6 expressed in Xenopus oocytes is significantly suppressed by activation of PKC-delta, which also altered its surface expression, while unaffected by PKA activation. SLC26A6 was also remarkably suppressed (with reduced surface and total protein expression) when WNK4 was co-expressed in oocytes. A closely related anion transporter, Pendrin (SLC26A4), was unaffected by PKC or WNK4. Based on these findings, the following specific aims will be pursued:1- Confirm that PKC-delta is the PKC isozyme mediating PKC regulation of SLC26A6 by testing the effects of PKC-delta specific peptide activator and inhibitor, and by evaluating whether PKC-delta translocates from cytosol to membrane. Identify the domain(s) of SLC26A6 essential for its regulation by PKC by analysis of SLC26A6-Pendrin chimeric transporters. 2- Confirm the findings in oocytes by assessing the effects of PKC activation on activity and surface localization of the endogenously expressed SLC26A6 in MPT cells (a mouse proximal tubule cell line). Confirm that PKC-delta is also the involved PKC isoform. Confirm any domain (s) that is (are) identified as critical for PKC regulation of SLC26A6 activity in oocytes in these cells. 3- Evaluate whether WNK4 suppressive regulation of SLC26A6 is at a translational or a post-translational level, such as enhanced endocytic retrieval and subsequent targeting for degradation. Identify the domains of SLC26A6 required for its regulation by WNK4 using a similar approach as in Aim 1.4- Use the domains of SLC26A6 mediating its regulation by PKC and WNK4 to identify potential associated proteins by the yeast-2-hybrid method. Confirm the association of identified proteins with SLC26A6 by co-precipitation experiments. Evaluate the functional roles of these proteins in mediating regulation of SLC26A6 by PKC and WNK4.
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