Abstract
Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
Highlights
Rapamycin had no impact on Big-conductance Ca2+-activated K+ (BK) channel mRNA levels (Figures 1E,F) and there was no difference in BK channel protein levels between the control and rapamycin treatment groups (Figures 1A–D)
We investigated Mechanistic target of rapamycin (mTOR) complex modulation of BK channels in podocytes and found novel modulation by mTORC2 of BK channels in podocytes via two different pathways (Figure 8)
One pathway is through mTORC2 regulation of the distribution of BK channels through Akt and the other is through mTORC2 regulation of BK protein expression through SGK1 in podocytes
Summary
Big-conductance Ca2+-activated K+ (BK) channels, which couple intracellular electrical and chemical signals, are found in most excitable, as well as non-excitable cells, and play important roles in a variety of physiological functions, including regulation of neuronal firing, endocrine cell secretion, smooth muscle tone, and cellular proliferation and migration (Contreras et al, 2013; Pantazis and Olcese, 2016; Latorre et al, 2017; Maqoud et al, 2017; Dopico et al, 2018).mTORC2 Regulates BK ChannelsIn the renal system, BK channels are highly expressed in nephrons and are important in K+ handling (Estilo et al, 2008). Some podocyte proteins functionally related to the foot process cytoskeleton, such as nephrin, Nep, the nephrin-like protein, and transient receptor potential cation channels, such as TRPC6, have been found to interact with BK channels directly and regulate the physiology and pathology of podocytes (Kim et al, 2008, 2009a,b, 2010). Abnormal expression of BK channels on the podocyte cell surface in some pathological conditions causes the destruction of cell actin cytoskeleton integrity, indicating that BK channels are essential for modulating the podocyte cytoskeleton (Kim and Dryer, 2011; Piwkowska et al, 2015). BK channels are important in Ca2+ homeostasis in podocytes and influence glomerular filtration
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