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. 2008 Jul;295(1):F235-46.
doi: 10.1152/ajprenal.00140.2008. Epub 2008 May 14.

Nephrin binds to the COOH terminus of a large-conductance Ca2+-activated K+ channel isoform and regulates its expression on the cell surface

Eun Young Kim  1 , Kyoung-Jae Choi, Stuart E Dryer
Affiliations

Nephrin binds to the COOH terminus of a large-conductance Ca2+-activated K+ channel isoform and regulates its expression on the cell surface

Eun Young Kim et al. Am J Physiol Renal Physiol. 2008 Jul.

Abstract

We carried out a yeast two-hybrid screen to identify proteins that interact with large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene. Nephrin, an essential adhesion and scaffolding molecule expressed in podocytes, emerged in this screen. The Slo1-nephrin interaction was confirmed by coimmunoprecipitation from the brain and kidney, from HEK-293T cells expressing both proteins, and by glutathione S-transferase pull-down assays. We detected nephrin binding to the Slo1 VEDEC splice variant, which is typically retained in intracellular stores, and to the beta4-subunit. However, we did not detect significant binding of nephrin to the Slo1 QEERL or Slo1 EMVYR splice variants. Coexpression of nephrin with Slo1 VEDEC increased expression of functional BKCa channels on the surface of HEK-293T cells but did not affect steady-state surface expression of the other COOH-terminal Slo1 variants. Nephrin did not affect the kinetics or voltage dependence of channel activation in HEK-293T cells expressing Slo1. Stimulation of Slo1 VEDEC surface expression in HEK-293T cells was also observed by coexpressing a small construct encoding only the distal COOH-terminal domains of nephrin that interact with Slo1. Reduction of endogenous nephrin expression by application of small interfering RNA to differentiated cells of an immortalized podocyte cell line markedly reduced the steady-state surface expression of Slo1 as assessed by electrophysiology and cell-surface biotinylation assays. Nephrin therefore plays a role in organizing the surface expression of ion channel proteins in podocytes and may play a role in outside-in signaling to allow podocytes to adapt to mechanical or neurohumoral stimuli originating in neighboring cells.

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Figures

Fig. 1.
Fig. 1.
Nephrin binds to large-conductance Ca2+-activated K+ (BKCa) channel subunits. A: coimmunoprecipitation of nephrin observed by immunoblot analysis after immunoprecipitation using antibodies against Slo1 or the β4-subunit of BKCa channels from mouse kidney or mouse brain, as indicated. The “input” lane contained a diluted sample of cell extract used to illustrate electrophoretic mobility; it was not designed for quantification of signal intensity. B: coimmunoprecipitation of Slo1 observed by immunoblot analysis after immunoprecipitation with an antibody against nephrin (top). This procedure was carried out on HEK-293T cells transiently expressing Myc-tagged isoforms Slo1VEDEC, Slo1QEERL, or Slo1EMVYR, as indicated, and channels were detected using anti-Myc. Note that a strong signal was only detected from cells expressing the Slo1VEDEC isoform. Interaction with Slo1EMVYR was detectable but much weaker. IB analysis shows that the various Slo1 variants (middle) and nephrin (bottom) were expressed at comparable levels throughout the assay. C: glutathione S-transferase (GST) pull-down assay showing that a GST fusion protein containing the COOH-terminal sequences of Slo1VEDEC could bind to nephrin, whereas GST and a GST fusion protein containing the COOH-terminal sequences of Slo1QEERL did not. The interaction with Slo1EMVYR with nephrin was very weak. D: confocal microscopy showing colocalization of endogenous Slo1 and nephrin in differentiated cells of a podocyte cell line (top) and in HEK-293T cells transiently expressing nephrin and Slo1VEDEC (bottom). Middle, control images from podocytes in which the primary antibody was a species-appropriate IgG. E: immunoblot using the same commercial antibody against nephrin on extracts of differentiated and undifferentiated cells from a podocyte cell line. IB, immunoblot; IP, immunoprecipitation.
Fig. 2.
Fig. 2.
Coexpression of nephrin with Slo1VEDEC increases steady-state expression on the surface of HEK-293T cells. A: confocal immunofluorescence using antibodies against the Myc tags of transiently expressed Slo1VEDEC channels. Cell surface channels in cells expressing Myc-tagged Slo1VEDEC were labeled with an FITC-conjugated goat anti-Myc applied to intact cells (green). After fixation and permeabilization, intracellular channels were stained using a mouse anti-Myc revealed using Alexa568-conjugated anti-mouse IgG (red). Identical laser excitation intensities and detection sensitivities were used for image collection from all of these samples. B: representative cell surface biotinylation assay shows that nephrin coexpression caused marked increase in surface expression of Slo1VEDEC but had no effect on Slo1QEERL or Slo1EMVYR, which had high levels of constitutive surface expression even in the absence of nephrin. C: densitometric quantification of 3 repetitions of the experiment shown in B. Data are means ± SE of relative Slo1 expression in the absence (C) or presence (N) of nephrin.
Fig. 3.
Fig. 3.
Effects of nephrin coexpression on whole cell currents recorded from HEK-293T cells expressing various Slo1 splice variants. Recording electrodes contained 5 μM free Ca2+ to allow for activation of BKCa channels by families of depolarizing voltage steps (shown in A) from a holding potential of −60 mV. A: representative traces illustrating a marked increase of currents in cells coexpressing nephrin with Slo1VEDEC. Nephrin had no effect in cells expressing the other isoforms, which exhibited large currents even in the absence nephrin. B: quantification of results from many cells. Data are means ± SE (n > 15 cells in each group). The only significant effect of nephrin was in cells expressing Slo1VEDEC (P < 0.05).
Fig. 4.
Fig. 4.
Nephrin does not substantially alter gating properties of BKCa channels. A: representative traces of whole cell currents evoked by depolarizing steps to +80 mV from a holding potential of −60 mV in HEK-293T cells expressing different Slo1 isoforms, as indicated, in the absence (solid traces) or presence (shaded traces) of nephrin. Amplitudes of traces are scaled to allow comparison of their time courses. Recording electrodes contained 5 μM Ca2+. B: activation curves constructed from HEK-293T cells expressing Slo1VEDEC in the presence and absence of nephrin. Nephrin markedly increased mean current at all membrane potentials but did not produce any substantial effects on the voltage dependence of activation.
Fig. 5.
Fig. 5.
Differentiated cells from a podocyte cell line express functional BKCa channels. A: immunoblot analysis using isoform-specific antibodies showing expression of Slo1VEDEC and Slo1QEERL in differentiated cells of a podocyte cell line. Adjacent lines are extracts of HEK-293T cells expressing the corresponding Slo1 isoform. The antibodies used in this analysis were described previously and do not cross-react (16). B: confocal analysis using the same antibodies also shows that both isoforms were expressed in differentiated podocytes. Note signal in paranuclear intracellular compartments and in foot processes. Control images are from cells in which rabbit IgG was used as primary antibody. C: endogenous BKCa currents in podocytes revealed by whole cell recordings using microelectrodes containing 5 μM free Ca2+. Note very slow activation kinetics and partial blockade after treatment with 500 nM iberiotoxin (IbTX) and complete blockade after 1 μM paxilline. Bar graph at right shows means ± SE for repetitions of this experiment (n > 10 cells per group). Mean currents in all 3 groups are significantly (P < 0.05) different from each other, as determined using one-way ANOVA and Tukey's honest significant difference test for unequal sample size.
Fig. 6.
Fig. 6.
Nephrin is required for normal surface expression of BKCa channels in differentiated cells of a podocyte cell line. A: immunoblot analysis showing that treatment with a small interfering (si)RNA directed against nephrin markedly reduced expression of nephrin but had no effect on total expression of NEPH1 in podocytes. A control siRNA had no effect. B: application of nephrin siRNA reduced surface expression of Slo1 channels as determined by cell surface biotinylation assay. Representative blots are shown at top, and densitometric analyses of 3 repetitions of this experiment are shown at bottom. C: representative traces illustrating that nephrin siRNA caused marked reduction in BKCa currents measured using whole cell recordings as Fig. 5. D: means ± SE of BKCa currents (n > 15 cells per group). The means are significantly different (P < 0.05), as determined using Student's unpaired t-test.
Fig. 7.
Fig. 7.
Evidence that the distal COOH-terminal portion of nephrin binds to Slo1VEDEC. Composition of a series GST-fusion proteins is indicated above a representative GST pull-down assay showing binding of the nephrin-CT2 construct (composed of residues R1160–V1241) to Slo1VEDEC. The assay was carried out on lysates of HEK-293T cells expressing this Slo1 splice variant.
Fig. 8.
Fig. 8.
Coexpression of a green fluorescent protein (GFP) fusion protein containing only the distal COOH-terminal residues of nephrin (GFP-nephrin-CT2) causes an increase in surface expression of Slo1VEDEC in HEK-293T cells but is not as effective as full-length nephrin. A: colocalization of the GFP-nephrin-CT2 with Myc-tagged Slo1VEDEC (red) as revealed using confocal microscopy. Note expression of both proteins on the cell surface and accumulation in perinuclear regions. B: coexpression of GFP-nephrin-CT2 caused a marked increase in steady-state surface expression of Slo1VEDEC as determined using cell surface biotinylation assays. Representative blots (top) and quantitative densitometric analysis of 3 repetitions of this experiment (bottom) are shown. Control cells expressed GFP. C: coexpression of GFP-nephrin-CT2 caused a statistically significant (P < 0.05) increase in mean whole cell currents in cells expressing Slo1VEDEC. Control cells coexpressed GFP.
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