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

Seung-Kuy Cha,Chunfa Huang,Yaxian Ding,Xiaoping Qi,Chou-Long Huang,R Tyler Miller

doi:10.1074/jbc.m110.160390

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 Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(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 Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.

Highlights

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]
We found previously that the Ca2؉-sensing receptor (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
The first is regulation of activity through control of cell surface expression via a mechanism dependent on G␣q and caveolin-1; the second is demonstration of physiologic regulation of Kir4.1; and the third feature is that the effect of the CaR on distal nephron NaCl transport appears to be attributable to physiologic regulation of a basolateral channel

Summary

EXPERIMENTAL PROCEDURES

All chemicals were purchased from Sigma or Fisher Scientific unless specified otherwise. Transfection, Immunoblotting, and Immunoprecipitation— HEK-293 cells were transiently transfected with the CaRWT and CaR mutants (CaRI40F, CaRL125P, and CaRR795W), and the epitope-tagged channel constructs indicated (Myc-tagged Kir4.1-pCruz and HA-tagged Kir4.1pCruz) or ROMK-pcDNA3.1 using the FuGENE 6 reagent and incubated at 37 °C for 24 – 48 h. The cells were lysed with 1ϫ radioimmune precipitation assay buffer, and the lysates were centrifuged at 15,000 rpm for 1 h at 4 °C. The antibody-loaded Dynabead-protein A complex was rinsed twice, and the beads were mixed with the various cell lysates and rotated in the cold room overnight. The samples were heated at 55 °C for 20 min for channel proteins or boiled for 5 min for the CaR and subjected to SDS-PAGE for immunoblotting using the antibodies indicated. The agarosebound complexes were precipitated and size-fractionated by SDS-PAGE, and proteins were identified by immunoblotting [17]

RESULTS

DISCUSSION

Tyler Miller