Atrial natriuretic peptide (ANP) responds to an elevated vascular volume by signaling an increase in glomerular filtration rate and Na excretion. The mechanism for the altered glomerular hemodynamics is partly due to the fact that, in glomerular mesangial cells (MC), ANP stimulates the cGMP-PKG pathway which activates large, Ca-activated K channels (BK) and hyperpolarizes the membrane potential rendering MC hyporesponsive to contractile agonists. PKG-1 appears to specifically (distinct from PKA and PKC) and directly activate BK in MC and vascular smooth muscle. However, in brain and other tissues, BK are regulated by PKA or indirectly by PKG which activates a protein phosphatase. BK are comprised of an alpha subunit (which is encoded by the slopoke gene; hSLO-alpha) with conserved membrane spanning properties similar to other voltage-gated K channels and an accessory beta sub7unit with a structure that differs substantially depending on the tissue of origin. Evidence shows that the beta subunit can account for many of the tissue-dependent differences in Ca sensitivity and other biophysical and regulatory properties of BK. The advent of a recently developed Mbeta1 knockout mouse permits us to determine if BK-beta is important for the sensitivity of BK to PKG with an important role to modulate mesangial tone and GFR.. These mice are significantly hypertensive, consistent with the view of dysfunctional regulation of BK by Ca and ANP/cGMP/PKG in vascular smooth muscle and glomerular mesangial cells. Functionally and structurally, the mouse BK-beta (Mbeta1) is very similar to the human Bkbeta (Hbeta2). Our preliminary data shows: 1. Hbeta2 and Mbeta1, when co-expressed with hSlo-alpha in HEK293 cells, confers the sensitivity of BK to Ca and activation by the cGMP pathway. 2. Beta-antisense oligonucleotides render the Ca sensitivity of mesangial BK to that found for the hSlo-alpha expressed in HEK293. 3. Western blot analysis shows that ANPO causes the translocation of PKG1 to the plasma membrane. 4. The GFR and Na excretion rates of volume expanded Mbeta1 knockout mice are less than 40% of the values for wild type controls. Therefore, it is hypothesized that, although the hSlo-alpha subunit is phosphorylated by PKG, the beta subunit is necessary for the ANP- and cGMP-evoked activation of BK. This pathway partly contributes to an elevated GFR in response to a volume load. The following Specific Aims will utilize both in vitro (cell cultures) and in vivo -(beta knockout) experimental models; #1. Determine if Hbeta2 confers regulation of BK by cGMP activated kinase 1 (PGK1). #2. Determine the mechanism of phosphorylating BK by PKG. #3. Determine the physiological significance of the BK-beta subunit in ANP-and cGMP-evoke activation of BK in glomerular mesangial cells. Regulation of BK via the cGMP-dependent pathway may be critical for vascular tone and renal maintenance of volume balance. Ultimately, better management of some forms of hypertension could develop from an in-depth understanding of BK regulatory mechanism at the molecular level.
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