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Caroline R Sussman,
Jinhua Zhao,
Consuelo Plata, Jing Lu,
Christopher Daly,
Nathan Angle,
Jennifer DiPiero,
Iain A Drummond,
Jennifer O Liang,
Walter F Boron,
Michael F Romero,
Min-Hwang Chang
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ABSTRACT: Mutations in the electrogenic Na+/nHCO3- cotransporter (NBCe1, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCe1 (zNBCe1) is 80% identical to published mammalian NBCe1 cDNAs. Like other fish NBCe1 clones, zebrafish NBCe1 is most similar to the pancreatic form of mammalian NBC (Slc4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential (Vm), voltage clamp (VC), intracellular pH (pH(i)), or intracellular Na+ activity (aNa(i)), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic Na+ and HCO3- transport as well as similar drug sensitivity, including inhibition by 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and tenidap. These data indicate that NBCe1 in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of Na+ and HCO3-. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology.
AJP Cell Physiology 08/2009; 297(4):C865-75. · 3.54 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 06/2007; 292(5):C1787-98. · 3.54 Impact Factor
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ABSTRACT: 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.
AJP Cell Physiology 11/2006; 291(4):C788-801. · 3.54 Impact Factor
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ABSTRACT: Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 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% CO2 (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.
Journal of Biological Chemistry 08/2006; 281(28):19241-50. · 4.77 Impact Factor
