Calcium-sensing Receptor Decreases Cell Surface Expression of the Inwardly Rectifying K+ Channel Kir4.1

Abstract

The Ca 2؉ -sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K ؉ channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K ؉ conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated G␣ q reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via G␣ i and G␣ q . Other ␣ subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of G␣ q on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K ؉ channel, an apical membrane distal nephron K ؉ channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves G␣ q and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.Sodium transport in the distal nephron determines body volume and blood pressure as demonstrated by the fact that mutations in genes expressed in this region of the kidney can cause either salt retention and high blood pressure or salt wasting and low blood pressure (1). A number of genetically distinct syndromes caused by abnormalities of transport proteins expressed in the distal nephron lead to phenotypes characterized by salt wasting. Bartter syndrome can result from loss of function mutations of the NaK 2 Cl co-transporter (NKCC2), 2 the renal outer medullary K ϩ channel (ROMK or Kir1.1), or the ClC-Kb (basolateral Cl Ϫ channel). A variant also characterized by sensorineural deafness is caused by loss of function mutations in an accessory protein for ClC-Kb, barttin (Bartter syndrome neuraosensory deafness or BSND) (1-5). The hypocalciuric, hypomagnesemic variant of Bartter syndrome, Gitelman syndrome, is due to loss of function mutations of the thiazide-sensitive NaCl co-transporter (6). The recently described SeSAME (seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance) or EAST (epilepsy, ataxia, sensorineural deafness, and tubulopathy) syndrome is caused by loss of function mutations in Kir4.1 (KCNJ10), an inwardly rectifying K ϩ channel that is expressed in the basolateral membrane of the distal nephron (7-10). Presumably, reduced acti...

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

“…8,9 This 40 pS K + channel is a heterotetramer of Kir4.1 and Kir5.1 because the coexpression of Kir4.1/5.1 produced an inwardly rectifying K + channel with the same biophysical properties as the 40 pS K + channel expressed in the basolateral membrane of the native DCT. [10][11][12] The role of Kir4.1 in forming the basolateral 40 pS K + channel of the DCT was convincingly demonstrated by the observation that the disruption of Kcnj10 (Kir.4.1) completely eliminated the 40 pS K + channel. 13 Moreover, the basolateral K + conductance was largely abolished in the early DCT (DCT1) of Kcnj10 2/2 mice, suggesting that Kcnj10 (Kir.4.1) is a major contributor to the basolateral K + conductance in the DCT1.…”

“…12,19,36 Also, it has been shown that cav-1 interacts directly with c-Src and the interaction domain is within residues 82-101. 37 Therefore, it is possible that cav-1 provides a microenvironment for the interaction between SFK and Kir.4.1.…”

“…This possibility is further enhanced by the observation that Kcnj10 interacted with CaSR and cav-1 and was inhibited by CaSR activation. 12 However, further study is required to address the role of CaSR in decreasing the basolateral K + conductance of the DCT in cav-1 KO mice. Figure 11 is a cell scheme illustrating the mechanism by which the disruption of cav-1 impairs the transport in the DCT.…”

Genetics of Magnesium Disorders

“…K ir 4.1 mutation interferes with basolateral K + recycling, compromising Na-K-ATPase function and generation of the apical membrane potential. Convincing evidence has shown that CaSR, coexpressed in the same region, inhibits cell surface expression of K ir 4.1 [70] via a mechanism dependent on G q and caveolin-1 [70,71] . Reduced Na-KATPase activity due to K ir 4.1 mutations causes a reduced apical NCC activity and apical membrane voltage, the major driving force for Mg 2+ influx [72] .…”

“…Reduced Na-KATPase activity due to K ir 4.1 mutations causes a reduced apical NCC activity and apical membrane voltage, the major driving force for Mg 2+ influx [72] . Importantly, CaSR is able to modulate K ir 4.1-mediated potassium extrusion in response to the physiological range of the extracellular Ca 2+ concentration (EC 50 Ca 2+ of 1.0 m M ) [70] . Thus, through CaSR, K ir 4.1 regulates the distal nephron NaCl transport and apical membrane potential physiologically.…”