Kir5.1 regulates Kir4.2 expression and is a key component of the 50-pS inwardly rectifying potassium channel in basolateral membrane of mouse proximal tubules

Abstract

Kir5.1 encoded by Kcnj16 is an inwardly rectifying K⁺ channel subunit, and it possibly interacts with Kir4.2 subunit encoded by Kcnj15 for assembling a Kir4.2/Kir5.1 heterotetramer in the basolateral membrane of mouse proximal tubule. We now used patch clamp technique to examine basolateral K⁺ channels of mouse proximal tubule (PT) and an immunoblotting/immunofluorescence (IF) staining microscope to examine Kir4.2 expression in wild-type and Kir5.1-knockout mice. IF staining shows that Kir4.2 was exclusively expressed in the proximal tubule, whereas Kir5.1 was expressed in the proximal tubule and distal nephrons including distal convoluted tubule. Immunoblotting showed that the expression of Kir4.2 monomer was lower in Kir5.1-knockout mice than that in the wild-type mice. In contrast, Kir4.1 monomer expression was increased in Kir5.1 knockout mice. IF images further demonstrated that the basolateral membrane staining of Kir4.2 was significantly decreased in Kir5.1 knockout mice. This is in sharp contrast to Kir4.1, which also interacts with Kir5.1 in the distal nephron, and IF images show that Kir4.1 membrane expression was still visible and unchanged in Kir5.1 knockout mice. The single channel recording detected a 50-pS inwardly rectifying K⁺ channel, presumably a Kir4.2/Kir5.1 heterotetramer, in the basolateral membrane of the proximal tubule of Kir5.1 wild-type mice. However, this 50-pS K⁺ channel was completely absent in the basolateral membrane of the proximal tubule of Kir5.1 knockout mice. Moreover, the membrane potential of the proximal tubule was less negative in Kir5.1 knockout mice than wild-type mice. We conclude that Kir5.1 is essential for assembling basolateral 50-pS K⁺ channel in proximal tubule and that deletion of Kir5.1 decreased Kir4.2 expression in the proximal tubule thereby decreasing the basolateral K⁺ conductance and the membrane potentials.NEW & NOTEWORTHY Our study provides direct evidence for the notion that Kir5.1 is a key component of a 50-60 pS inwardly-rectifying-K⁺ channel, a main type K⁺ channel in the basolateral-membrane of PT. Also, we demonstrate that deletion of Kir5.1 decreased Kir4.2 protein expression including the basolateral-membrane in PT. Finally, depolarization of PT-membrane- potential in Kir5.1-knockout mice suggests that Kir4.2 alone is not able to sustain basolateral K⁺ conductance of the PT in the absence of Kir5.1.

Keywords: Kcnj10; Kcnj15; Kcnj16; potassium channel; proximal tubule.

Fig. 1. Kir5.1 is expressed in the proximal tubule.

(A) Immunofluorescence (IF) staining of Kir5.1 in the wild-type (A-a, b, c) and Kir5.1-KO mice (A-d & e) (LTL staining was used as a marker of proximal tubule. Enlarged immunofluorescence images show Kir5.1-staining in LTL-positive proximal tubule (A-b) and in LTL-negative tubules (A-c), respectively. (B) Double-IF staining shows the expression of Kir5.1 (red), ATP1a1 (green) and the both (merge) in the cortex (B-a, b, c) and in the medulla (B-d, e, f) of wild-type mice. An overlap between Kir5.1 and ATP1a1 is indicated by an arrow.

Fig.2.. Kir4.2 membrane staining is decreased in Kir5.1-KO mice.

Immunofluorescence staining of Kir4.2 in the proximal tubule of the wild-type with low magnification (A) and with high magnification (B). Immunofluorescence staining of Kir4.2 in the proximal tubule of Kir5.1-KO mice with low magnification (C) and with high magnification (D). LTL staining was used as a marker of proximal tubule.

Fig. 3. Deletion of Kir5.1 decreased Kir4.2 expression.

(A) A western blot shows the expression of Kir4.2, Kir4.1, ROMK and Kir5.1 in wild-type and in Kir5.1-KO mice. (B) A bar-graph with a scatter plot shows normalized band density of Kir4.2, Kir4.1 and ROMK. An arrow indicates the band of Kir4.2 monomer, Kir4.1 monomer and ROMK monomer used for the normalized calibration. ROMK band corresponds to a glycosylated ROMK monomer. Kir4.1 immunoblot shows a major band around 220-kDa which is Kir4.1/Kir5.1 heterotetramer in the wild-type mice. Although this band is also present in Kir5.1-KO mice, it is Kir4.1 homotetramer rather than Kir4.1/Kir5.1 heterotetramer because this band is absent with Kir5.1 antibody.

Fig.4. Deletion of Kir5.1 decreased Kir4.2 basolateral membrane expression.

Immunofluorescence staining of Kir4.2 (red), ATP1a1 (green) and both (merge) in wild-type mice (A-C) and in Kir5.1-KO mice (D-F). An overlap between Kir4.2 and ATP1a1 is indicated by an arrow. (E ) A bar graph with scatter plot summarizes the relative fluorescence-signaling-intensity ratio between Kir4.2 and ATP1a1. Fluorescence intensity was measured with Zenblue 3.3 edition software provided by Zeiss laser scanning microscope (LSM) 980 plus Airyscan 2 confocal microscope.

Fig. 5. Deletion of Kir5.1 has no effect on basolateral membrane expression of Kir4.1.

(A) Immunofluorescence staining of Kir4.1 in the LTL-negative distal tubules of the wild-type mice (A-a) or in Kir5.1-KO mice (A-b). (B) Double-immunofluorescence staining shows the expression of Kir4.1 (red), ATP1a1 (green) and the both (merge) in wild-type mice (B-a, b, c) and in Kir5.1-KO mice (C-a, b, c). An overlap between Kir4.1 and ATP1a1 is indicated by an arrow. (C ) A bar graph with scatter plot summarizes the relative fluorescence-signaling-intensity ratio between Kir4.1 and ATP1a1.

Fig.6. A 50-pS K⁺ channel activity is absent in the basolateral membrane of the proximal tubule of Kir5.1-KO mice.

(A) A single channel recording made in a cell-attached patch shows the 50-pS K⁺ channel activity in the basolateral membrane of proximal tubule of wild-type mice. The membrane holding potentials are indicated on the top of each trace and channel close line is indicated by a dotted line and a letter of C. The pipette solution contains (in mM) 140 KCl, 2 MgCl₂, 1 EGTA and 10 HEPES and the bath solution contains 135 NaCl, 5 KCl, 2 MgCl₂, 1.8 CaCl₂, 5 mM glucose and 5 HEPES. (B) The channel current-voltage curve yields a slope conductance of 50-pS between −20 to −60 mV. (C) A scatter plot shows voltage-dependent estimated channel open probability. (D) A table summarizes the probability of finding the 50-pS K⁺ channel in the basolateral membrane of proximal tubule from wild-type and Kir5.1-KO mice (Data are obtained from both male and female mice).

Fig. 7. Deletion of Kir5.1 depolarizes the membrane potential of proximal tubules.

A whole-cell recording shows I-reversal potential of the proximal tubule measured in the wild-type mice (A) and in Kir5.1-KO mice (B). A combined recording shows that I-reversal-potentials of the proximal tubule in Kir5.1-KO mice is shifted to right (depolarization) in comparison to the wild-type mice (C) (Two traces are different from A &B). The results are summarized in a bar graph with scatter plot (D) illustrating the individual value of the I-reversal-potential obtained in male and female wild-type and Kir5.1-KO mice.