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. 2012 Jul;23(7):1172-80.
doi: 10.1681/ASN.2011100972. Epub 2012 Apr 19.

Protein kinase G inhibits flow-induced Ca2+ entry into collecting duct cells

Juan Du  1 , Wei-Yan Wong, Lei Sun, Yu Huang, Xiaoqiang Yao
Affiliations

Protein kinase G inhibits flow-induced Ca2+ entry into collecting duct cells

Juan Du et al. J Am Soc Nephrol. 2012 Jul.

Abstract

The renal cortical collecting duct (CCD) contributes to the maintenance of K(+) homeostasis by modulating renal K(+) secretion. Cytosolic Ca(2+) ([Ca(2+)](i)) mediates flow-induced K(+) secretion in the CCD, but the mechanisms regulating flow-induced Ca(2+) entry into renal epithelial cells are not well understood. Here, we found that atrial natriuretic peptide, nitric oxide, and cyclic guanosine monophosphate (cGMP) act through protein kinase G (PKG) to inhibit flow-induced increases in [Ca(2+)](i) in M1-CCD cells. Coimmunoprecipitation, double immunostaining, and functional studies identified heteromeric TRPV4-P2 channels as the mediators of flow-induced Ca(2+) entry into M1-CCD cells and HEK293 cells that were coexpressed with both TRPV4 and TRPP2. In these HEK293 cells, introducing point mutations at two putative PKG phosphorylation sites on TRPP2 abolished the ability of cGMP to inhibit flow-induced Ca(2+) entry. In addition, treating M1-CCD cells with fusion peptides that compete with the endogenous PKG phosphorylation sites on TRPP2 also abolished the cGMP-mediated inhibition of the flow-induced Ca(2+) entry. Taken together, these data suggest that heteromeric TRPV4-P2 channels mediate the flow-induced entry of Ca(2+) into collecting duct cells. Furthermore, substances such as atrial natriuretic peptide and nitric oxide, which increase cGMP, abrogate flow-induced Ca(2+) entry through PKG-mediated inhibition of these channels.

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Figures

Figure 1.
Figure 1.
Effect of cGMP, PKG, atrial natriuretic peptide, and nitric oxide on flow-induced Ca2+ entry in M1-CCD cells. (A, C, and E) Representative traces illustrating the time course of flow-induced [Ca2+]i responses at a shear stress of (A and C) 5 dyne/cm2 or (E) 0.5 dyne/cm2 (low saline solution [SS]). (B, D, and F) Summary showing the maximal change in flow-induced [Ca2+]i responses (Δ[Ca2+]i) for cells at a shear stress of (B and D) 5 dyne/cm2 or (F) 0.5 dyne/cm2 (low SS). Cells were pretreated for 10 minutes with (A, B, E, and F) 2 mM 8-Br-cGMP with or without 1 µM KT5823, (C, D, and F) 10 nM atrial natriuretic peptide (ANP) with or without 1 µM KT5823, or (C, D, and F) 50 µM SNAP with or without 1 µM KT5823. Control had no treatment. Cells were bathed in NPSS containing 1% BSA except for 0PSS series (a bar in B), in which the bath was Ca2+-free. The solid bar on top of the traces indicates the period when laminar flow was applied. Data are given as the mean ± SE (n=8–10 experiments and 10–20 cells per experiment). **P<0.01 versus 8-Br-cGMP (B and F). *P<0.01 versus control (D and F). ##P<0.01 versus the corresponding ones without KT5823. OPSS, Ca2+-free physiological saline solution.
Figure 2.
Figure 2.
TRPV4 and TRPP2 involvement in flow-induced [Ca2+]i entry in M1-CCD cells. (A and C) Representative traces illustrating the time course of flow-induced [Ca2+]i responses. (B and D) Summary showing the maximal change in flow-induced [Ca2+]i responses (Δ[Ca2+]i). In A and B, the cells were transfected with an empty plasmid vector (vector), TRPV4M680D (V4M680D), or TRPP2D511V (P2D511V). In C and D, the cells were pretreated with Preimmune IgG (Preimmu) or T1E3. Data are given as the mean ± SE (n=8–10 experiments and 10–20 cells per experiment). ΔP<0.01 versus vector.
Figure 3.
Figure 3.
Physical interaction between TRPV4 and TRPP2 in M1-CCD cells. (A and B) Coimmunoprecipitation. The pulling antibody and the blotting antibody are indicated. Control immunoprecipitation was performed using preimmune IgG (labeled as Preimmu). Anti-P2 indicates anti-TRPP2, anti-V4 indicates anti-TRPV4, IB indicates immunoblot, and IP indicates immunoprecipitation (n=3 experiments). (C–D″) Double immunostaining. (C and D) TRPV4 and (C′ and D′) TRPP2 were colocalized in (C″ and D″; merge) the cytosol, the plasma membrane, and the primary cilium of M1-CCD cells. D–D″ are confocal images of z-series stacks showing the primary cilium emerging from the apical membrane.
Figure 4.
Figure 4.
Physical association and functional role of TRPV4 and TRPP2 in HEK293 cells that were coexpressed with TRPV4 and TRPP2. (A and B) Flow-induced [Ca2+]i responses. (A) Representative traces illustrating the time course of flow-induced [Ca2+]i responses. (B) Summary showing the maximal change in flow-induced [Ca2+]i responses (Δ[Ca2+]i). Cells were bathed in NPSS containing 1% BSA except for 0PSS series (labeled bars in B), in which the bath was Ca2+-free. Cells were transfected with an empty vector (vector), TRPP2 (P2), TRPV4 (V4), TRPV4+TRPP2 (V4+P2), TRPV4M680D+TRPP2 (V4M680D+P2), or TRPV4+TRPP2D511V (V4+P2D511V). Data are given as the mean ± SE (n=6–8 experiments and 8–20 cells per experiment). ΔP<0.01 versus vector. +P<0.01 versus V4+P2. (C and D) Coimmunoprecipitation. The pulling antibody and the blotting antibody are indicated. Control immunoprecipitation was performed using the preimmune IgG (Preimmu; n=3 experiments). (E–F″) Double immunostaining. (E and F) TRPV4 and (E′ and F′) TRPP2 were colocalized in (E″ and F″; merge) the cytosol, the plasma membrane, and the primary cilium of transfected HEK293 cells. F–F″ are confocal images of z-series stacks showing the primary cilium emerging from the apical membrane.
Figure 5.
Figure 5.
Flow-induced [Ca2+]i responses in HEK293 cells that were transfected with individual TRPV4 or TRPP2 or cotransfected with both TRP isoforms. (A and C) Representative traces illustrating the time course of flow-induced [Ca2+]i responses. (B, D, and E) Summary showing the maximal change in flow-induced [Ca2+]i responses. HEK293 cells stably transfected with a PKG1α gene are labeled as PHEK cells. All cells were bathed in NPSS containing 1% BSA. Cells were pretreated with 8-Br-cGMP (2 mM) with or without KT5823 (1 µM) for 10 minutes. Data are given as the mean ± SE (n=6–8 experiments and 8–20 cells per experiment). V4 indicates TRPV4, P2 indicates TRPP2, and P2S827A and P2T719A are point mutants of TRPP2, respectively. ◊P<0.01 compared with vector. ψP<0.01 compared with 8-Br-cGMP alone. ∞P<0.01 compared with V4+P2 without 8-Br-cGMP. &P<0.01 compared with V4+P2 with 8-Br-cGMP.
Figure 6.
Figure 6.
PKG phosphorylation sites in M1-CCD cells. (A) Representative traces illustrating the time course of flow-induced [Ca2+]i responses. (B) Summary showing the maximal change in flow-induced [Ca2+]i responses (Δ[Ca2+]i). All cells were bathed in NPSS containing 1% BSA. Cells were pretreated with 8-Br-cGMP (2 mM) with or without TAT-P2T719 (100 nM) and/or TAT-P2S827 (100 nM) for 10 minutes before flow. Data are given as the mean ± SE (n=6–8 experiments and 8–20 cells per experiment). ΦP<0.01 versus 8-Br-cGMP.
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References

    1. Giebisch G: Renal potassium transport: Mechanisms and regulation. Am J Physiol 274: F817–F833, 1998 - PubMed
    1. Liu W, Morimoto T, Woda C, Kleyman TR, Satlin LM: Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct. Am J Physiol Renal Physiol 293: F227–F235, 2007 - PubMed
    1. Satlin LM, Sheng S, Woda CB, Kleyman TR: Epithelial Na(+) channels are regulated by flow. Am J Physiol Renal Physiol 280: F1010–F1018, 2001 - PubMed
    1. Zeidel ML: Renal actions of atrial natriuretic peptide: Regulation of collecting duct sodium and water transport. Annu Rev Physiol 52: 747–759, 1990 - PubMed
    1. Ortiz PA, Garvin JL: Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol 282: F777–F784, 2002 - PubMed

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