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. 2010 Sep;299(3):F594-604.
doi: 10.1152/ajprenal.00206.2010. Epub 2010 Jul 14.

Regulation of podocyte BK(Ca) channels by synaptopodin, Rho, and actin microfilaments

Eun Young Kim  1 , Jae Mi Suh, Yu-Hsin Chiu, Stuart E Dryer
Affiliations

Regulation of podocyte BK(Ca) channels by synaptopodin, Rho, and actin microfilaments

Eun Young Kim et al. Am J Physiol Renal Physiol. 2010 Sep.

Abstract

Mechanosensitive large-conductance Ca(2+)-activated K(+) channels encoded by the Slo1 gene (BK(Ca) channels) are expressed in podocytes. Here we show that BK(Ca) channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with BK(Ca) channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of BK(Ca) channels to the cell surface. Moreover, addition of exogenous beta-actin to mouse podocyte lysates reduces BK(Ca)-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of BK(Ca) channels in HEK293T cells. However, Synpo does not affect the stability of cell surface BK(Ca) channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect BK(Ca) gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of BK(Ca) channels but does not affect total expression of BK(Ca) channels or their gating. The effects of Synpo on surface expression of BK(Ca) are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface BK(Ca) channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate BK(Ca) channels through its effects on actin dynamics and by modulating interactions between BK(Ca) channels and regulatory proteins of the podocyte slit diaphragm.

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Figures

Fig. 1.
Fig. 1.
Interaction between pore-forming subunit of large-conductance Ca2+-activated K+ (BKCa) channels (Slo1) and synaptopodin (Synpo). A: representative example of reciprocal coimmunoprecipitation of Slo1 and Synpo transiently expressed in HEK293T cells. Immunoprecipitation (IP) and detection were carried out with antibodies against the Flag tag on Synpo and against the Myc tag on Slo1. In controls, immunoprecipitation was carried out with IgG. In this and subsequent figures, a sample of the cell lysate was used as a positive control, and these lanes are marked “Input.” IB, immunoblot. B: reciprocal coimmunoprecipitation of Slo1 and Synpo in differentiated cells of a mouse podocyte cell line using an antibody against Synpo carried and a custom-made anti-Slo1. C: reciprocal coimmunoprecipitation of Slo1 and Synpo in differentiated cells of a mouse podocyte cell line using anti-Synpo and a commercially available anti-Slo1 (see materials and methods).
Fig. 2.
Fig. 2.
Colocalization and interaction of Slo1 and Synpo in mouse glomeruli. A: from a paraffin section of mouse kidney cortex, confocal image of double-label immunofluorescence showing Slo1 (red), Synpo (green), and a merged image, as indicated. Note colocalization of Synpo and Slo1 along the surface of the glomerular basement membrane, indicating expression in podocyte foot processes. There also appears to be Slo1 expression in locations consistent with expression in mesangial cells. B: reciprocal coimmunoprecipitation of Slo1 and Synpo in isolated mouse glomeruli.
Fig. 3.
Fig. 3.
Interactions between Slo1 and Synpo mapped with glutathione S-transferase (GST) pull-down assays. A: Synpo was pulled out of podocyte lysates (arrow) with a GST fusion protein comprised of the most distal portion of the large cytosolic COOH terminal of Slo1 (CT3) but not by GST or the other GST-Slo1 fusion proteins described below. B: mapping of Synpo interactions using 4 smaller GST-Slo1 fusion proteins that span the distal portion of the COOH terminal. CT3B contains an actin-binding domain described previously (37). C: adding recombinant β-actin to podocyte lysates causes concentration-dependent reduction in interaction between Synpo and GST-Slo1 fusion protein.
Fig. 4.
Fig. 4.
Coexpression of Synpo increases steady-state surface expression of Slo1 in HEK293T cells. A: representative cell surface biotinylation assay in HEK293T cells transiently expressing Myc-Slo1 and/or Flag-Synpo as indicated. Stimulatory effect of Synpo was blocked in cells treated with a membrane-permeant C3 transferase (C3T), a Rho inhibitor. Coexpression of Synpo did not affect total expression of Slo1 relative to actin. B: densitometric quantification of 4 repetitions of this experiment presented as means ± SE.
Fig. 5.
Fig. 5.
Coexpression of Synpo increases macroscopic currents through BKCa channels in HEK293T cells transiently expressing Slo1. A: representative traces showing families of whole cell currents evoked by depolarizing steps as indicated above the current traces. B: current-voltage diagram showing that currents were increased at all step potentials. In this and subsequent panels, numbers of cells in each group (n) are indicated. C: normalization of currents to the average evoked by steps to +80 mV (G/G80) reveals that coexpression of Synpo does not affect the voltage dependence of BKCa activation. D: Synpo coexpression does not affect BKCa activation kinetics. y-Axis shows mean time constant (τ) fitted to rising phase of macroscopic currents evoked by steps to +40, +60, and +80 mV. None of the differences is statistically significant (n.s.). E: time course of internalization of cell surface Slo1 proteins in HEK293T cells expressing Slo1 by itself (●) or coexpressing Synpo (■). Data points represent means ± SE of 4 repetitions of this experiment. OD492 is optical density at 492 nm. Superimposed curve shows exponential decay with a time constant of 16 h.
Fig. 6.
Fig. 6.
Characteristics of podocyte cell lines. Controls are an immortalized mouse podocyte cell line, whereas Synpo KD is a similar line containing a stably incorporated small interfering RNA that targets Synpo expression. A: immunoblot analyses show that the Synpo KD line has markedly reduced expression of Synpo, but this does not affect expression of total actin or total Slo1. Cont, control. B: confocal images show that Synpo KD lines have almost complete loss of Synpo and that this is associated with a reduction in the number of stress fibers seen with rhodamine-phalloidin staining.
Fig. 7.
Fig. 7.
Synpo and Slo1 distribution in control and Synpo KD podocyte cell lines. A: in control cells, there is close colocalization between Slo1 and Synpo in intracellular compartments and around the periphery of the cells, especially in regions of membrane ruffling. B: Synpo cannot be detected in Synpo KD cell lines, and there is a marked reduction in Slo1 expression in the periphery of the cells.
Fig. 8.
Fig. 8.
Synpo knockdown reduces cell surface expression of podocyte BKCa channels. A: representative cell surface biotinylation assay showing marked reduction in surface Slo1 in Synpo KD cells. Con, control. B: densitometric quantification of 3 replications of the cell surface biotinylation assay. Bar graphs show means ± SE. C: representative whole cell recordings made with recording pipettes containing 5 μM free Ca2+. In previous studies we have shown that all of these currents are blocked by the BKCa inhibitor paxiline (18). Note lower-amplitude currents in the recording from the Synpo KD cell. D: means ± SE of currents evoked by depolarizing step to +60 mV in control and Synpo KD cells. Numbers of cells are indicated above bars. *P < 0.05 (Student's unpaired t-test).
Fig. 9.
Fig. 9.
Rho inhibition reduces surface expression of BKCa channels in podocytes. A: representative pull-down assay showing Rho activation in control and Synpo KD podocytes treated with vehicle (Con) or cell-permeant C3 transferase (C3T), an inhibitor of Rho. Note that CT3 causes reduction in total and active Rho in control and Synpo KD cells and results in almost complete loss of active Rho in Synpo KD cells. B: confocal images showing distribution of Slo1 and F-actin (rhodamine-phalloidin) in podocyte cell lines treated with vehicle or C3T. Note disruption of actin filaments and reduction in surface expression of Slo1 as a result of C3T. C: mean ± SE currents evoked by depolarizing steps to +60 mV in whole cell recordings from podocytes treated with vehicle or C3T for 2 h. As with analyses of active Rho, the effect of C3T on current amplitude is quantitatively similar to effect of Synpo knockdown. *P < 0.05 compared with control; **P < 0.05 compared with Synpo KD cells treated with vehicle (1-way ANOVA followed by Tukey's post hoc test).
Fig. 10.
Fig. 10.
Sustained disruption of actin filaments reduces macroscopic BKCa recorded from podocyte cell lines. A: mean ± SE currents in control podocytes and in podocytes treated with cytochalasin D (Cyto D) for 15 min or 2 h as indicated. Only the effect of 2 h of Cyto D is significantly different from control (1-way ANOVA followed by Tukey's post hoc test). B: Cyto D treatment for 15 min is sufficient to disrupt actin filament organization in podocytes.
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