Caveolin-1 Deficiency Inhibits the Basolateral K⁺ Channels in the Distal Convoluted Tubule and Impairs Renal K⁺ and Mg²⁺ Transport

cav-1 is required for c-Src–induced stimulation of Kcnj10. (A) A set of recordings showing K⁺ currents measured with the perforated whole-cell recording in HEK293T cells transfected with KCNJ10, KCNJ10+c-Src, KCNJ10+cav-1, and KCNJ10+cav-1+c-Src. The pipette solution and bath solution contained a symmetrical 140 mM KCl, and the K⁺ currents were measured from –60 to 60 mV at 20-mV steps (the protocol of the voltage clamp is included). (B) Western blots showing the expression of KCNJ10, c-Src, and cav-1 (indicated by an arrow) in HEK293T cells.

cav-1 is required for the effect of SFK inhibitor on Kcnj10. (A) A bar graph summarizing the results of experiments in which Ba²⁺-sensitive K⁺ currents were measured at –60 mV with perforated whole-cell recording in the cells transfected with KCNJ10. (B) A set of recordings showing K⁺ currents measured with the perforated whole-cell recording in KCNJ10 or KCNJ10+cav-1–transfected HEK293T cells under control conditions (no PP1) or treated with 1 μM PP1. (C) A bar graph summarizing the results of experiments in which the effect of 1 μM PP1 on Ba²⁺-sensitive K⁺ currents was examined at –60 mV with perforated whole-cell recording in the cells transfected with Kcnj10/Kcnj16 or Kcnj10/Kcnj16+cav-1. (D) A bar graph summarizing the results of experiments in which Ba²⁺-sensitive K⁺ currents were measured at –60 mV with perforated whole-cell recording in the cells transfected with KCNJ10 (control), KCNJ10+cav-1, KCNJ10+WNK4, and KCNJ10+WNK4+cav-1.

Stimulatory effect of c-Src on Kcnj10^Y9F is diminished. (A) A set of recordings showing K⁺ currents measured with the perforated whole-cell recording in HEK293T cells transfected with KCNJ10^Y9F, KCNJ10^Y9F+c-Src, KCNJ10^Y9F+cav-1, and KCNJ10^Y9F+cav-1+c-Src. (B) A bar graph summarizing the results of experiments in which Ba²⁺-sensitive K⁺ currents were measured at –60 mV with perforated whole-cell recording in the cells transfected with KCNJ10 or KCNJ10^Y9F.

Lack of cav-1 reduced the basolateral K⁺ channel activity in the DCT. (A) A channel recording showing the 40 pS K⁺ channel activity of the DCT1 in cav-1 KO mice. The experiments were performed in a cell-attached patch, and the membrane holding potentials were indicated on the top of each trace. The channel closed level is indicated by a dotted line. (B) The mean channel open probability of the 40 pS K⁺ channel in DCT1 of WT or KO mice. (C) A bar graph showing the probability of finding the 40 pS K⁺ channel in the DCT1 of cav-1 KO mice. Black and gray bars represent empty patches (No K⁺ channel) and the patches with 40 pS K⁺ channel. (D) The mean channel numbers per patch in the DCT1 of WT and cav-1 KO mice, respectively. (E) Fluorescence microscope image showing the expression of Kcnj10 at a low magnification in the kidney of WT and cav-1 KO mice, respectively. (F) Kcnj10 immunostaining with a large magnification in the renal cortex of WT and cav-1 KO mice. A bar represents the size of 10 μm. The immunostaining was performed under identical conditions, and the kidney slices of both WT and KO mice were placed in the same slide.

Disruption of cav-1 decreased basolateral K⁺ conductance in DCT1. (A) Whole-cell patch recording demonstrating the effect of PP1, an inhibitor of SFK, on the K⁺ currents of the DCT1 in the WT and cav-1 KO mice, respectively. The pipette solution and bath solution contained a symmetrical 140 mM KCl, and the K⁺ currents were measured from –60 to 60 mV at 20-mV steps (the protocol of the voltage clamp is included). (B) A bar graph summarizing the results of experiments in which Ba²⁺-sensitive K⁺ currents in the DCT1 of WT or cav-1 KO mice were measured at –60 mV with perforated whole-cell recording in the presence of or in the absence of SFK inhibitor (1 μΜ PP1). The pipette and bath solution were the same as previously described. (C) K⁺ reversal potentials in the DCT1 of WT or cav-1 KO mice were measured with perforated whole-cell recording in the presence of or in the absence of SFK inhibitor (1 μΜ PP1). The DCT1 was bathed in the 140 mM Na⁺/5 mM K⁺ bath solution, and the pipette solution contained 140 mM KCl.

Lack of cav-1 inhibits the basolateral Cl^– channels in DCT1 and the expression of SPAK and NCC. (A) A recording showing NPPB-sensitive Cl^– currents in DCT1 of WT and cav-1 KO mice. The measurements were carried out with perforated whole-cell recording with symmetrical 140 mM K⁺ in the bath and pipette. (B) The results from five experiments measured at –60 mV are summarized in a bar graph. (C) A Western blot showing the expression of full-length SPAK (F-SPAK) in WT and cav-1 KO mice. (D) A Western blot showing the expression of Thr⁵³ phosphorylated NCC (pNCC) and total NCC in the WT and cav-1 KO mice, respectively.

Disruption of cav-1 does not inhibit the transcription of NCC. (A) Fluorescence microscope image at a low magnification showing the expression of NCC in the kidney of WT and cav-1 KO mice, respectively. (B) NCC immunostaining with a high magnification in the renal cortex of WT and cav-1 KO mice. A bar represents the size of 5 μm. The immunostaining was performed under identical conditions, and the kidney slices of both WT and KO mice were placed in the same slide. (C) Agarose gel showing the band of (105bp) NCC, (150bp) full-length SPAK (F-SPAK), and (138bp) kidney-specific SPAK (Ks-SPAK), respectively. (D) A bar graph summarizing the corresponding results. (E) A Western blot using lysates from renal cortex demonstrating the expression of TRPV5 in WT and cav-1 KO mice.

Disruption of cav-1 increases the expression of ENaC-α and ENaC-β. (A) A recording showing K reversal potentials in the CNT of WT or cav-1 KO mice. The experiments were performed with perforated whole-cell recording with 5 mM K⁺ in the bath and 140 mM K⁺ in the pipette. (B) A Western blot showing the expression of ENaC-α and ENaC-β in WT and cav-1 KO mice, respectively. (C) Fluorescence microscope image showing the expression of ENaC-α in the renal cortex and outer medulla (OM) of WT and cav-1 KO mice, respectively. (D) Fluorescence microscope image showing the expression of ENaC-β in the renal cortex and OM of WT and cav-1 KO mice, respectively. A bar represents the size of 10 μm. The immunostaining was performed under identical conditions, and the kidney slices of both WT and KO mice were placed in the same slide.

cav-1 KO mice are hypomagnesemic, hypocalcemic, and hypokalemic. A bar graph showing the mean plasma Na⁺, Ca²⁺, Mg²⁺, and K⁺ concentrations in the WT and cav-1 KO mice (n=4), respectively.