Biomedical subjects

Angelito A Bernardo

Publications and source records attributed to Angelito A Bernardo.

3 recordsLinked to original sources

PubMed2006

Role of NH(2) and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1.

Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH(2) (DeltaN424) or the entire COOH (DeltaC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. DeltaN424 and DeltaC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the DeltaN424, DeltaC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total DeltaC92 expression in the cell was constant. We found that decreased DeltaC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both DeltaN424 and DeltaC92 were cotransfected together and more stable expression of DeltaC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH(2) epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane.

Amino Acid Sequence↗

PubMed2006

The sodium bicarbonate cotransporter: structure, function, and regulation.

The role of the Na(+)-coupled HCO(3)(-) transporter (NBC) family is indispensable in acid-base homeostasis. Almost all tissues express a member of the NBC family. NBC has been studied extensively in the kidney and plays a role in proximal tubule HCO(3)(-) reabsorption. Although the exact function of this transporter family on other tissues is not very clear, the ubiquitous expression of NBC family suggests a role in cell pH regulation. Altered NBC activity caused by mutations of the gene responsible for NBC protein expression results in pathophysiologic conditions. Mutations of NBC resulting in important clinical disorders have been reported extensively on one member of the NBC family, the kidney NBC (NBC1). These mutations have led to several structural studies to understand the mechanism of the abnormal NBC1 activity.

Acidosis, Renal Tubular↗

PubMed2002

A central role for Pyk2-Src interaction in coupling diverse stimuli to increased epithelial NBC activity.

Regulation of renal Na-HCO cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO(2)) requires both Src family kinase (SFK) and classic MAPK pathway activation. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) couples discrete G protein-coupled receptor and growth factor receptor signaling to SFK activation. We examined the role of Pyk2-SFK interaction in coupling these stimuli to increased NBC1 activity in opossum kidney cells. Carbachol increased tyrosine autophosphorylation of endogenous Pyk2 and ectopically expressed wild-type Pyk2 and were abrogated by kinase-dead mutant (Pyk2-KD) overexpression. Pyk2 phosphorylation was calcium/calmodulin dependent, and Pyk2 associated with Src by means of SH2 domain interaction. Pyk2 phosphorylation and Pyk2-Src interaction by carbachol were mimicked by both ANG II and CO(2). To correlate Pyk2 autophosphorylation and Pyk2-Src interaction with NBC activity, cotransporter activity was measured in untransfected cells and in cells overexpressing Pyk2-KD in the presence or absence of carbachol, ANG II, or CO(2). In Pyk2-KD-overexpressing cells, the effect of carbachol, ANG II, and CO(2) was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO(2) to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2-Src interaction.

Acidosis↗