Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO 3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I NBC ) by two-electrode voltage clamp (Day 3), injected CA II protein ؉ Tris or just Tris (Day 3), measured I NBC or the initial rate at which the intracellular pH fell (dpH i /dt) upon applying 5% CO 2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I NBC or dpH i /dt (Day 4). Because dpH i /dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I NBC slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5 end of NBCe1-A and CA II at the 3 end (EGFP-e1-CAII). We measured I NBC or dpH i /dt (days 3-4), exposed oocytes to EZA, and measured I NBC or dpH i /dt (Day 3-4). dpH i /dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.The electrogenic Na/bicarbonate cotransporter (NBCe1 or e1) 3 plays a central role in HCO 3 Ϫ reabsorption and regulation of intracellular pH (1). The kidney-specific splice variant NBCe1-A is localized at the basolateral membrane of renal proximal tubule cells (2), where it mediates efflux of HCO 3 Ϫ (and/or CO 3 ϭ ). Cytoplasmic HCO 3 Ϫ arises from the intracellular hydration of CO 2 , which is catalyzed by the cytoplasmic enzyme carbonic anhydrase II (CA II) (3).Because the reports of Vince and Reithmeier (4 -6) that cytosolic CA II binds to the LDADD motif on the cytoplasmic C terminus of the Cl-HCO 3 exchanger AE1, Sterling et al. (7,8) have measured rates of intracellular pH (pH i ) change in HEK293 cells transiently transfected with AE1 and concluded that CA II enhances AE1-mediated HCO 3 Ϫ transport. The C termini of all three NBCe1 splice variants, i.e. NBCe1-A as well as the more universally expressed variant NBCe1-B (9, 10) and the brain-specific NBCe1-C (11), have two motifs similar to LDADD in AE1. Moreover, isothermal titration calorimetry and pulldown assays suggest that, at least under non-reducing conditions, the common C terminus of NBCe1-A/B interacts with CA II in vitro (12). Similarly, Pushkin and co-workers (12-15) working with a mouse proximal convoluted tubule (mPCT) cell line stably transfected with NBCe1-A, concluded that CA II enhances the current carried by NBCe1-A.For three reasons, we set out to verify the hypothesis that CA II enhances the activity of NBCe1-A. First, a pH i measurement, as in the AE1 study, is an indirect index of the rate of HCO 3 Ϫ ...

The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na(+) affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

Others have shown that H(2)DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO(3) exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (-160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK (wt, approximately 40 microM) < NKMIK congruent with NKMIN congruent with KKMIN < KNMIN congruent with KNMIK < NNMIK < NNMIN ( approximately 400 microM) < DDMID < EEMIE ( approximately 800 microM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80-91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

During apoptosis, dying cells are quickly internalized by neighboring cells or phagocytes, and are enclosed in phagosomes that undergo a maturation process to generate the phagoslysosome, in which cell corpses are eventually degraded. It is not well understood how apoptotic cell degradation is regulated. Here we report the identification and characterization of the C. elegans tbc-2 gene, which is required for the efficient degradation of cell corpses. tbc-2 encodes a Rab GTPase activating protein (GAP) and its loss of function affects several events of phagosome maturation, including RAB-5 release, phosphatidylinositol 3-phosphate dynamics, phagosomal acidification, RAB-7 recruitment and lysosome incorporation, which leads to many persistent cell corpses at various developmental stages. Intriguingly, the persistent cell corpse phenotype of tbc-2 mutants can be suppressed by reducing gene expression of rab-5, and overexpression of a GTP-locked RAB-5 caused similar defects in phagosome maturation and cell corpse degradation. We propose that TBC-2 functions as a GAP to cycle RAB-5 from an active GTP-bound to an inactive GDP-bound state, which is required for maintaining RAB-5 dynamics on phagosomes and serves as a switch for the progression of phagosome maturation.

BackgroundPreoperative differentiation of borderline from malignant epithelial ovarian tumors (BEOT from MEOT) can impact surgical management. MRI has improved this assessment but subjective interpretation by radiologists may lead to inconsistent results.PurposeTo develop and validate an objective MRI‐based machine‐learning (ML) assessment model for differentiating BEOT from MEOT, and compare the performance against radiologists' interpretation.Study TypeRetrospective study of eight clinical centers.PopulationIn all, 501 women with histopathologically‐confirmed BEOT (n = 165) or MEOT (n = 336) from 2010 to 2018 were enrolled. Three cohorts were constructed: a training cohort (n = 250), an internal validation cohort (n = 92), and an external validation cohort (n = 159).Field Strength/SequencePreoperative MRI within 2 weeks of surgery. Single‐ and multiparameter (MP) machine‐learning assessment models were built utilizing the following four MRI sequences: T2‐weighted imaging (T2WI), fat saturation (FS), diffusion‐weighted imaging (DWI), apparent diffusion coefficient (ADC), and contrast‐enhanced (CE)‐T1WI.AssessmentDiagnostic performance of the models was assessed for both whole tumor (WT) and solid tumor (ST) components. Assessment of the performance of the model in discriminating BEOT vs. early‐stage MEOT was made. Six radiologists of varying experience also interpreted the MR images.Statistical TestsMann–Whitney U‐test: significance of the clinical characteristics; chi‐square test: difference of label; DeLong test: difference of receiver operating characteristic (ROC).ResultsThe MP‐ST model performed better than the MP‐WT model for both the internal validation cohort (area under the curve [AUC] = 0.932 vs. 0.917) and external validation cohort (AUC = 0.902 vs. 0.767). The model showed capability in discriminating BEOT vs. early‐stage MEOT, with AUCs of 0.909 and 0.920, respectively. Radiologist performance was considerably poorer than both the internal (mean AUC = 0.792; range, 0.679–0.924) and external (mean AUC = 0.797; range, 0.744–0.867) validation cohorts.Data ConclusionPerformance of the MRI‐based ML model was robust and superior to subjective assessment of radiologists. If our approach can be implemented in clinical practice, improved preoperative prediction could potentially lead to preserved ovarian function and fertility for some women.Level of EvidenceLevel 4.Technical EfficacyStage 2. J. Magn. Reson. Imaging 2020;52:897–904.