Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases

Abstract

Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na+/H+ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca2+, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca2+, and an active phorbol ester. These experiments indicate that Na+/H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.

Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro.

“…The latter result contrasted with results of Weinman and co-workers (27), who demonstrated that calcium-calmodulin protein kinase II inhibited the activity ofthe reconstituted renal brush border Na+-H+ exchanger. The reasons for the differences observed are unclear but may be related to differences in preparations (intact tubule vs. reconstituted Na+-H+ exchanger).…”

Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(+)-H+ exchanger.

“…The cDNA encodes a protein consisting of 358 amino acids with a calculated molecular mass of 38.6 kD. This predicted size is less than that suggested from prior studies using column chromatography and autoradiography of SDS-PAGE gels containing the cofactor phosphorylated by PKA (4)(5)(6)(7)12). To clarify this issue, cell-free translation studies were performed.…”

“…RT-PCR was performed initially using rabbit kidney RNA and oligonucleotide or random primers for reverse transcription. For the PCR reaction, the oligonucleotide sense primer, 5 '-GAAGCGCAGCAGCAGACGGGC-3', and antisense primer, 5 '-ACTGGAGAATCGGGCGGAGAGG-3', were used. These primers spanned the stop codon region of the cDNA.…”

“…In vitro studies involving solubilized membranes and a reconstitution assay have suggested that the inhibitory effect of PKA on the activity of the transporter requires participation of another intermediary protein; a regulatory cofactor (NHE-RF) (5)(6)(7). A partial amino acid sequence of this putative cofactor, from which an affinity-purified polyclonal antibody to an artificial peptide representing NHE-RF was developed, has been obtained (7).…”

Defective Parathyroid Hormone Regulation of NHE3 Activity and Phosphate Adaptation in Cultured NHERF-1-/- Renal Proximal Tubule Cells

“…In the present studies, for reasons not immediately apparent, the response to PTH in NHERF-1 Ϫ/Ϫ proximal tubule cells was variable and, in fact, some of the cultures showed significant stimulation of NHE3 activity. It is worth noting that some prior studies have suggested that the direct effect of activation of protein kinase C is to activate NHE3 (39). In this regard then, NHERF-1 knockout mice may prove valuable in sorting out potential regulatory processes affecting the relation between PTH receptor activation and NHE3 activity.…”