Abstract
Kcnj10 encodes the inwardly rectifying K(+) channel Kir4.1 in the basolateral membrane of the distal convoluted tubule (DCT) and is activated by c-Src. However, the regulation and function of this K(+) channel are incompletely characterized. Here, patch-clamp experiments in Kcnj10-transfected HEK293 cells demonstrated that c-Src-induced stimulation of Kcnj10 requires coexpression of caveolin-1 (cav-1), and immunostaining showed expression of cav-1 in the basolateral membrane of parvalbumin-positive DCT. Patch-clamp experiments detected a 40-pS inwardly rectifying K(+) channel, a heterotetramer of Kir4.1/Kir5.1, in the basolateral membrane of the early DCT (DCT1) in both wild-type (WT) and cav-1-knockout (KO) mice. However, the activity of this basolateral 40-pS K(+) channel was lower in KO mice than in WT mice. Moreover, the K(+) reversal potential (an indication of membrane potential) was less negative in the DCT1 of KO mice than in the DCT1 of WT mice. Western blot analysis demonstrated that cav-1 deficiency decreased the expression of the Na(+)/Cl(-) cotransporter and Ste20-proline-alanine-rich kinase (SPAK) but increased the expression of epithelial Na(+) channel-α. Furthermore, the urinary excretion of Mg(2+) and K(+) was significantly higher in KO mice than in WT mice, and KO mice developed hypomagnesemia, hypocalcemia, and hypokalemia. We conclude that disruption of cav-1 decreases basolateral K(+) channel activity and depolarizes the cell membrane potential in the DCT1 at least in part by suppressing the stimulatory effect of c-Src on Kcnj10. Furthermore, the decrease in Kcnj10 and Na(+)/Cl(-) cotransporter expression induced by cav-1 deficiency may underlie the compromised renal transport of Mg(2+), Ca(2+), and K(+).
Highlights
The transport of NaCl in the Distal convoluted tubule (DCT) is a two-step process: NaCl enters the cell through apical Na-Cl cotransporter (NCC), and Na+ leaves the basolateral membrane by Na-K-ATPase while Cl– leaves the cells through the basolateral Cl– channels or KCl cotranspoter.[3,4,5]
Inhibition of SFK has been shown to decrease the basolateral membrane of DCT,[13] it is reasonable to assume that Kir4.1 K+ channel activity in mouse DCT1.9 We examined and cav-1 are colocalized in the basolateral membrane of DCT whether cav-1 was required for the effect of PP1, an incells
We performed the perforated ylated Kcnj[10] and that the inhibition of src-family protein whole-cell recording in HEK293T cells transfected with tyrosine kinase (SFK), a family of c-Src-like tyrosine kinase,[22] decreased the basolateral 40 pS K+ channel activity in DCT1.9 we examined whether cav-1 was
Summary
In the CCD characterized by dotted staining (arrow) in the basolateral membrane. (E) An image with a high magnification showing the absence of cav-1 staining in the gl in. Cav-1 KO mice have a renal phenotype similar to the Gitelman’s syndrome, except plasma Ca2+
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