Analysis of the interaction between human kidney anion exchanger 1 and kanadaptin using yeast two-hybrid systems

Abstract Kidney anion exchanger adaptor protein (Kanadaptin) is a protein which interacts with the cytoplasmic N-terminal domain of kidney anion exchanger 1 (kAE1) and was first detected in mice using the yeast two-hybrid system and was also found to co-localize with kAE1 in rabbit a-intercalated cells. Impaired trafficking of human kAE1 can result in the kidney disease-distal renal tubular acidosis (dRTA), and defective interaction between human kAE1 and kanadaptin may cause this trafficking impairment and be the basis for dRTA pathogenesis. However, it is unknown whether kAE1 can really interact with kanadaptin in humans. We have thus investigated the interaction between human kAE1 and human kanadaptin by using both Gal4 and LexA yeast two-hybrid systems. It was found that co-expression of Gal4DBD fused to the cytoplasmic N-terminal domain of kAE1 and Gal4AD fused to kanadaptin could not activate the transcription of the ADE2, HIS3 and lacZ reporters in the Gal4 system. A similar result was obtained for the interaction between B42AD fused to the cytoplasmic N-terminal domain of kAE1 and LexA fused to kanadaptin in activation of lacZ transcription in the LexA system. The absence of interaction between the fusion proteins in both yeast two-hybrid systems raises the possibility that kAE1 may not interact with kanadaptin in human cells. Considerably different structures of both kAE1 and kanadaptin in mice and humans may lead to different binding properties of the proteins in these two species

Analysis of the interaction between human kidney anion exchanger 1 and kanadaptin using yeast two-hybrid systems

Kidney anion exchanger adaptor protein (Kanadaptin) is a protein which interacts with the cytoplasmic N-terminal domain of kidney anion exchanger 1 (kAE1) and was first detected in mice using the yeast two-hybrid system and was also found to co-localize with kAE1 in rabbit a-intercalated cells. Impaired trafficking of human kAE1 can result in the kidney disease-distal renal tubular acidosis (dRTA), and defective interaction between human kAE1 and kanadaptin may cause this trafficking impairment and be the basis for dRTA pathogenesis. However, it is unknown whether kAE1 can really interact with kanadaptin in humans. We have thus investigated the interaction between human kAE1 and human kanadaptin by using both Gal4 and LexA yeast two-hybrid systems. It was found that co-expression of Gal4DBD fused to the cytoplasmic N-terminal domain of kAE1 and Gal4AD fused to kanadaptin could not activate the transcription of the ADE2, HIS3 and lacZ reporters in the Gal4 system. A similar result was obtained for the interaction between B42AD fused to the cytoplasmic N-terminal domain of kAE1 and LexA fused to kanadaptin in activation of lacZ transcription in the LexA system. The absence of interaction between the fusion proteins in both yeast two-hybrid systems raises the possibility that kAE1 may not interact with kanadaptin in human cells. Considerably different structures of both kAE1 and kanadaptin in mice and humans may lead to different binding properties of the proteins in these two species

Dynamic Kv4.3–CaMKII unit in heart: an intrinsic negative regulator for CaMKII activation

Aims Reduction of transient outward current (Ito) and excessive activation of C

Regulated acid-base transport in the collecting duct

The renal collecting system serves the fine-tuning of renal acid-base secretion. Acid-secretory type-A intercalated cells secrete protons via a luminally expressed V-type H(+)-ATPase and generate new bicarbonate released by basolateral chloride/bicarbonate exchangers including the AE1 anion exchanger. Efficient proton secretion depends both on the presence of titratable acids (mainly phosphate) and the concomitant secretion of ammonia being titrated to ammonium. Collecting duct ammonium excretion requires the Rhesus protein RhCG as indicated by recent KO studies. Urinary acid secretion by type-A intercalated cells is strongly regulated by various factors among them acid-base status, angiotensin II and aldosterone, and the Calcium-sensing receptor. Moreover, urinary acidification by H(+)-ATPases is modulated indirectly by the activity of the epithelial sodium channel ENaC. Bicarbonate secretion is achieved by non-type-A intercalated cells characterized by the luminal expression of the chloride/bicarbonate exchanger pendrin. Pendrin activity is driven by H(+)-ATPases and may serve both bicarbonate excretion and chloride reabsorption. The activity and expression of pendrin is regulated by different factors including acid-base status, chloride delivery, and angiotensin II and may play a role in NaCl retention and blood pressure regulation. Finally, the relative abundance of type-A and non-type-A intercalated cells may be tightly regulated. Dysregulation of intercalated cell function or abundance causes various syndromes of distal renal tubular acidosis underlining the importance of these processes for acid-base homeostasis