| Large-conductance, Ca2+-activated K+ channels (BK) are composed of pore-forming BK-alpha subunits as well as modulatory BK-beta subunits. This thesis examined the localization and the functional role of the BK-beta1 subunit in two distinct cell types of the mammalian kidney---mesangial cells, and epithelial cells of the distal nephron. BK-beta1 was identified on the RNA and protein levels in cultured human mesangial cells (MC), smooth-muscle like cells that surround the filtration capillaries of the kidney glomeruli. To identify a putative role for BK-beta1 in MC, the kidney function (glomerular filtration rate, or GFR) of "knockout" (Mbeta1-/-) mice was compared to that of wild-type. Although euvolemic values were not different from those of wild-type, under conditions of acute volume expansion, GFR was significantly attenuated in Mbeta1-/-. These observations indicate that the beta1 subunit of BK is present in the mesangium and contributes to the increased GFR that accompanies an acute salt and volume load.; These studies have also identified a role for BK-beta1 in the kaliuretic response to volume expansion. Under euvolemic conditions, neither the urinary excretion of K+ nor the fractional excretion of K+ were different between wild-type and Mbeta1-/-. However, whereas wild-type mice exhibited a robust increase in K+ secretion with volume expansion, this response was significantly attenuated in Mbeta1-/-. In fact, Mbeta1-/- mice were unable to increase K+ excretion with increasing flow. Using immunohistochemistry of murine renal sections, BK-beta1 was colocalized to the connecting tubule (CNT). Similarly, BK-beta1 also localized to rabbit CNT, and the RNA sequence of rabbit CNT BK-beta1 was found to be identical to that of rabbit muscle. In order to identify an in vitro model in which to examine the mechanism responsible for BK-beta1-dependent BK-alpha activation, it was established that Madin-Darby Canine Kidney Cells (MDCK; a distal nephron cell line) express functional BK channels and apically express BK-alpha and BK-beta1. In conclusion, BK-beta1 is expressed in both MC and CNT, where it increases the sensitivity of BK-alpha to changes that occur with volume expansion. Furthermore, MDCK cells may be convenient models to study the mechanism of flow-mediated K+ secretion. |
