Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitation (IP) analysis of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in mCCDcl1 cells and mouse kidney. A: immunoblots of mCCDcl1 cell homogenates show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). B: immunoblots of mouse kidney homogenates also show enrichment of BKα, SK3, and IK1 in the TRPV4 immunoprecipitate (TRPV4) compared with the IgG control (IgG). Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously ((20), Fig. 1). CAV-1 (22 kD) protein band is demonstrated in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunoprecipitation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Endogenous immunoprecipitaton (IP) analysis of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney. Immunoblots of mCCDcl1 cell homogenates show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (A). Immunoblots of mouse kidney homogenates also show robust enrichment of TRPV4, BKα, SK3, and IK1 in the CAV-1 immunoprecipitate (CAV-1) compared with the IgG control (IgG) (B). Endogenous immunoprecipitaton of IK1 (C) or BKα (D) followed by blotting for CAV-1, TRPV4, and each of the KCa channels, in mCCDcl1 cells, showed enrichment of CAV-1, TRPV4, BKα, SK3, and BKα or IK1. Appropriate protein bands for TRPV4 (98 kD), SK3 (81 kD), IK1 (45 kD), and BKα (110 kD) were verified previously [(20) Fig. 1]. CAV-1 (22 kD) was verified in Fig. 1. Immunoprecipitation experiments were repeated three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Effect of caveolin-1 (CAV-1) knockdown on transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated (GSK101) intracellular Ca2+ ([Ca2+]i) response to blockers of TRPV4 and KCa channels. The normal peak [Ca2+]i response to TRPV4 activation (GSK101) is markedly reduced from 597 ± 45 nM (n = 70) to 231 ± 47 nM (n = 85) following CAV-1 siRNA treatment (A and B). Furthermore, the apparent initial rate of TRPV4-mediated Ca2+ influx was markedly depressed from 108 ± 27 nM/min (n = 70) in normal conditions to 42 ± 16 nM/min (n = 85) in the presence of CAV-1 siRNA (C), reflecting a markedly depressed level of TRPV4 activation. The subsequent panels demonstrate that the typical normal peak elevation in [Ca2+]i following TRPV4 activation is markedly reduced upon inhibition of each KCa channel following CAV-1 knockdown. Iberiotoxin (IbTX) treatment reduces the peak [Ca2+]i for BK inhibition from 227 ± 34 nM (n = 73) to 84 ± 33 nM (n = 59) after CAV-1 siRNA treatment (D and E). TRAM-34 treatment reduces the peak [Ca2+]i for IK1 inhibition from 312 ± 31 nM (n = 90) to 81 ± 12 nM (n = 95) after CAV-1 siRNA treatment (F and G). Apamin treatment reduces the peak [Ca2+]i for SK3 inhibition from 414 ± 60 nM (n = 72) to 196 ± 23 nM (n = 60) after CAV-1 siRNA treatment (H and I). Therefore, after CAV-1 siRNA treatment the response for each of the KCa channels to TRPV4 activation is markedly depressed. ***P < 0.001. BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Activation Assay, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Representative immunofluorescence images show colocalization of transient receptor potential cation channel subfamily V member 4 (TRPV4) with each of the KCa channels in collecting duct mCCDcl1 cells in mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for TRPV4 (TRPV4, green) and SK3 (SK3-ATTO-594, red) show strong colocalization as apparent in the merged image (Merged, yellow) (A). Similarly, immunostaining for TRPV4 (red) and BKα (green) (B) and for TRPV4 (red) and IK1 (green) (C) demonstrated noted colocalization in the merged images (Merged, yellow). Mouse kidney CCD immunostained for TRPV4 (TRPV4, green) and SK3 (SK3, ATTO-594, red) display noted colocalization in the merged images (Merged, yellow) (D). In a similar manner, immunostaining for TRPV4 (TRPV4, red) and BKα (BKα, green) (E) and for TRPV4 (TRPV4, red) and IK1 (IK1, green) (F) likewise demonstrated apparent colocalization in the merged images (Merged, yellow). Areas of more diffuse staining are also apparent in some cases, especially for IK1 (see F). Note that we verified the primary antibodies using blocking peptides in an earlier publication [(20) Figs. 1 and ​and4].4]. All the immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; SK, small conductance Ca2+-activated K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunofluorescence, Immunostaining, Staining, Blocking Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Caveolae facilitate TRPV4-mediated Ca 2+ signaling and the hierarchical activation of Ca 2+ -activated K + channels in K + -secreting renal collecting duct cells

doi: 10.1152/ajprenal.00076.2018

Figure Lengend Snippet: Immunofluorescence images demonstrates colocalization of caveolin-1 (CAV-1) with transient receptor potential cation channel subfamily V member 4 (TRPV4) and each of the KCa channels in mCCDcl1 cells and mouse kidney cortical collecting duct (CCD). mCCDcl1 cells immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) showed strong colocalization in the merged image (Merged, yellow) (A). Similarly, immunostaining for CAV-1 with each of the KCa channel demonstrated robust colocalization in the merged images as shown for CAV-1 (green) and SK3 (red) (B), CAV-1 (red) and BKα (green) (C), and CAV-1 (red) and IK1 (green) (D). Diffuse localization was also apparent, especially for CAV-1 and IK1 (D). Mouse kidney CCD immunostained for CAV-1 (CAV-1, green) and TRPV4 (TRPV4-ATTO-550, red) also displayed robust colocalization as shown in the merged image (Merged, yellow) (E). Similarly, immunostaining for CAV-1 and each of the KCa channels displayed noted colocalization in the merged images (Merged, yellow) for CAV-1 (green) and SK3 (red) (F), for CAV-1 (red) and BKα (green) (G), and for CAV-1 (green) and IK1 (red) (H). Areas of more diffuse localization were also apparent, especially for BKα and IK1 (see merged images in G and H). Immunolocalization experiments were repeated at least three times (n = 3). BK, large conductance Ca2+-activated K+ channel; IK, intermediate conductance channel; SK, small conductance K+ channel.

Article Snippet: The following primary antibodies were used: anti-CAV-1 (Thermo Fisher, cat. no. MA-3-600), anti-SK1 (Alomone, cat. no. APC-039), anti-SK3-ATTO-594 (Alomone, cat. no. APC-025-AR), anti-IK1 (Alomone, cat. no. ALM-051), anti-BKα (Alomone, cat. no. APC-151), and anti-TRPV4 (Alomone, cat. nos.

Techniques: Immunofluorescence, Immunostaining