Vacuolar H+-ATPase B1 Subunit Mutations that Cause Inherited Distal Renal Tubular Acidosis Affect Proton Pump Assembly and Trafficking in Inner Medullary Collecting Duct Cells

Yang, Qiongqiong; Li, Guangmu; Singh, Satish K.; Alexander, E. A.; Schwartz, John H.

doi:10.1681/asn.2005121277

Cited by 43 publications

(38 citation statements)

References 24 publications

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“…It has been shown that B1 constructs bearing the single amino acid mutations described in dRTA patients do not assemble into functional V-ATPase complexes (67). These mutated B1 subunits impair the plasma membrane trafficking and insertion of the V-ATPase, possibly by competing with wild-type holoenzymes for components of the vesicle-targeting machinery.…”

Section: Discussionmentioning

confidence: 99%

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice

Păunescu

Russo

Silva

et al. 2007

American Journal of Physiology-Renal Physiology

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Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice. Am J Physiol Renal Physiol 293: F1915-F1926, 2007. First published September 26, 2007; doi:10.1152/ajprenal.00160.2007.-Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar protonpumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1Ϫ/Ϫ mice than in B1ϩ/ϩ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1Ϫ/Ϫ mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28 -40% of normal) V-ATPase function is preserved in medullary ICs from B1Ϫ/Ϫ mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions. proton pump; immunofluorescence; pH homeostasis; urinary acidification; Atp6v1b1Ϫ/Ϫ mice THE MAIN MEDIATOR OF INTRACELLULAR organelle acidification in eukaryotic cells and of proton (H ϩ ) secretion along the distal renal nephron is the ubiquitous vacuolar proton-pumping ATPase (vacuolar, or V-type, H ϩ -ATPase, or V-ATPase). The V-ATPase is a complex enzyme, consisting of two large sectors or domains (V 0 , the transmembrane domain involved in H ϩ translocation, and V 1 , the cytosolic domain, responsible for hydrolyzing ATP), which together contain at least 13 distinct

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Section: Discussionmentioning

confidence: 99%

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice

Păunescu

Russo

Silva

et al. 2007

American Journal of Physiology-Renal Physiology

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“…The role of a particular isoform composition in the intracellular trafficking, location, and/or function of the V-ATPase is still poorly understood and is currently under investigation. Recent studies, performed in transfected kidney inner medullary collecting duct cells, showed that B1 constructs carrying the single amino acid mutations described in dRTA patients fail to assemble into functional V-ATPase complexes (62). It was suggested that mutated B1 subunits might impair the trafficking of V-ATPase by competing with wild-type subunitcontaining complexes for as yet unidentified components of the protein/vesicle targeting machinery.…”

Section: Discussionmentioning

confidence: 99%

Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

Silva

Shum

El-Annan

et al. 2007

American Journal of Physiology-Cell Physiology

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Ϫ/Ϫ mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1 Ϫ/Ϫ mice compared with B1 ϩ/ϩ . Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1 Ϫ/Ϫ mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1 Ϫ/Ϫ mice as in B1 ϩ/ϩ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1 Ϫ/Ϫ mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1 Ϫ/Ϫ mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. male reproductive tract; male fertility; luminal acidification; proton pump; vacuolar H ϩ ATPase SPERMATOZOA DEVELOP their ability to become motile and to fertilize an egg as they pass through the male excurrent ducts, which are composed of efferent ducts, epididymis, and vas deferens. Considerable changes in the composition of luminal fluid occur in the epididymis and include net water, Na ϩ , Cl Ϫ , and HCO 3 Ϫ reabsorption, K ϩ secretion, in addition to secretion and resorption of numerous proteins (2,17,18,30,36,37,58,59). In addition, the lumen of the epididymis and vas deferens is maintained acidic compared with blood, and has a low bicarbonate concentration (36, 37). Low pH and bicarbonate are required for sperm maturation, for the prevention of premature activation of acrosomal enzymes, and for maintaining sperm in a quiescent, immotile state during their maturation and storage in the epididymis and vas deferens (1,2,16,26,32). Thus a defect in the acidification capacity of epididymis and vas deferens might have important consequences on the maturation and storage of spermatozoa, and, consequently, on male fertility (45).In mammals, the epididymis is composed of one to three highly coiled tubules divided into several morphologically distinct regions, including the initial segments, caput, corpus, and cauda epididymidis. These regions are further segmented into intraregional segments, which all contribute to the establishment of a succession of microenvironments in which spermatozoa mature and are concentrated, transported, and stored (19,31,49). The composition of the luminal microenvironment is controlled by distinct epithelial cell types: principal cells, narrow cells and clear cells line the ent...

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“…By contrast, in humans harboring B1 mutations, B2 replacement does not appear to take place, which results in the development of dRTA. Whether or not B2 is able to assemble into the holoenzyme in the presence of deficient B1, or whether the mutated B1 subunit by itself impairs V-ATPase trafficking, as was shown in cell cultures (Yang et al, 2006), are questions that will require further investigation. Future follow-up studies will also be necessary to determine whether or not human males with B1 and/or a4 mutations will develop infertility.…”

Section: V-atpase Isoform Compensatory Functionmentioning

confidence: 99%

Regulation of luminal acidification in the male reproductive tract via cell–cell crosstalk

Shum

Silva

Brown

et al. 2009

Journal of Experimental Biology

119

101

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Vacuolar H+-ATPase B1 Subunit Mutations that Cause Inherited Distal Renal Tubular Acidosis Affect Proton Pump Assembly and Trafficking in Inner Medullary Collecting Duct Cells

Cited by 43 publications

References 24 publications

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice

Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

Regulation of luminal acidification in the male reproductive tract via cell–cell crosstalk

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